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US20030129176A1 - Atherosclerosis-associated genes - Google Patents

Atherosclerosis-associated genes Download PDF

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US20030129176A1
US20030129176A1 US10/219,664 US21966402A US2003129176A1 US 20030129176 A1 US20030129176 A1 US 20030129176A1 US 21966402 A US21966402 A US 21966402A US 2003129176 A1 US2003129176 A1 US 2003129176A1
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protein
cells
cdna
cdnas
nrml
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Karen Jones
Wayne Volkmuth
Michael Walker
Lynn Murry
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Incyte Corp
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Incyte Genomics Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to a combination of isolated cDNAs that are significantly co-expressed with one or more known atherosclerosis-associated genes.
  • the invention also relates to the use of the combination in the diagnosis, prognosis, treatment, and evaluation of therapies for disorders associated with atherosclerosis.
  • Atherosclerosis is a disorder characterized by cellular changes in the arterial intima and the formation of arterial plaques containing intracellular and extracellular deposits of lipids.
  • the thickening of artery walls and the narrowing of the arterial lumen underlies the pathologic condition in most cases of coronary artery disease, aortic aneurysm, peripheral vascular disease, and stroke.
  • a number of metabolic pathways and a cascade of molecular events is involved in the cellular morphogenesis, proliferation, and cellular migration that results in atherogenesis (Libby et al. (1997) Int J Cardiol 62 (S2):23-29).
  • the artery walls consist of three layers: the intima (innermost), the media, and the adventitia (outermost).
  • the intima consists of a layer of endothelial cells lining the lumen of arteries and arterioles. Endothelial cells form a barrier against the indiscriminate entry of substances from the blood into the artery. Specific transporter proteins expressed by endothelial cells facilitate barrier function. Endothelial cells also secrete a number of substances which help regulate downstream vascular contractility blood coagulation, and other aspects of vascular biology.
  • the medial layer of the arterial wall contains smooth muscle cells in a matrix of collagen and elastic fibers produced by the smooth muscle cells.
  • the outermost layer of the arterial wall, the adventitia, is a mixture of collagen bundles, elastic fibers, some smooth muscle cells, fibroblasts and nerve cells.
  • the adventitia provides structural integrity to the blood vessel and acts as a support matrix for the media and intima.
  • Initiation of an atherosclerotic lesion often occurs following vascular endothelial cell injury often attributable to hypertension, diabetes mellitus, hyperlipidemia, fluctuating shear stress, smoking, or transplant rejection.
  • Nitric oxide and superoxide anions are released and react to form cytodestructive peroxynitrite radicals that cause injury to the endothelium and myocytes of the intima and lead to expression of a variety of molecules that produce local and systemic effects.
  • mediators of inflammation such as cytokines, complement components, prostaglandins, and downstream transcription factors.
  • Such mediators promote monocyte infiltration of the vascular intima and lead to the upregulation of adhesion molecules which encourages attachment of the monocytes to the damaged endothelial cells.
  • components of the extracellular matrix including collagens, fibrinogens, and matrix Gla protein are induced and provide sites for monocyte attachment, and annexins, plasminogen activator inhibitor 1, and nitric oxide synthases are induced to counteract these effects.
  • Monocytes that infiltrate the lesion accumulate modified low density lipoprotein through scavenger receptors such as CD36 and macrophage scavenger receptor type I.
  • modified lipids is a factor in atherogenesis and is influenced by modifying enzymes such as lipoprotein lipase, carboxyl ester lipase, serum amyloid P component, LDL-receptor related protein, microsomal triglyceride transfer protein, and serum esterases such as paraoxonase.
  • Lipid metabolism is governed by cholesterol biosynthesis enzymes such as 3-hydroxy-3-methylglutaryl coenzyme A synthase, and products of the apolipoprotein genes. Modified lipid stabilization and accumulation is aided by perilipin and alpha-2-macroglobulin.
  • plaque consists of a mass of lipid-engorged monocytes and a lipid-rich necrotic core covered by a fibrous cap.
  • the gradual progression of plaque growth is punctuated by thrombus formation and leads to clinical symptoms such as unstable angina, myocardial infarction, or stroke.
  • Thrombus formation is initiated by episodic plaque rupture which exposes flowing blood to tissue factors, which induce coagulation, and collagen, which activates platelets.
  • ECM extracellular matrix components
  • cytoskeletal proteins such as calponin, myosin, desmin, and other gene products in the cells.
  • smooth muscle cells lose their contractility and become able to migrate from the media to the intima, to proliferate, and to secrete extracellular matrix components which contribute to arterial intimal thickening.
  • the present invention satisfies a need in the art by providing a combination comprising a plurality of cDNAs that are useful for diagnosis, prognosis, treatment, and evaluation of therapies for disorders associated with atherosclerosis.
  • the invention provides a combination of isolated cDNAs that are significantly co-expressed with one or more known atherosclerosis-associated genes.
  • the combination comprises the isolated cDNAs having the nucleic acid sequences of SEQ ID NOs:1-25 and the complements of SEQ ID NOs:1-25.
  • the combination is placed on a substrate.
  • the substrate is a microarray.
  • the invention also provides a method for using the combination to detect gene expression in a sample containing nucleic acids, the method comprising hybridizing the substrate containing the combination to the nucleic acids of the sample under conditions for formation of one or more hybridization complexes and detecting hybridization complex formation, wherein complex formation indicates gene expression in the sample.
  • the sample is from artery or is obtained during microsurgery to open a blocked artery.
  • complex formation is compared to standards and is diagnostic of a disorder associated with atherosclerosis.
  • the invention further provides a method of using a combination to screen a plurality of molecules to identify at least one ligand which specifically binds a cDNA of the combination, the method comprising combining the substrate containing the combination with molecules under conditions to allow specific binding; and detecting specific binding, thereby identifying a ligand which specifically binds at least one cDNA of the combination.
  • the molecules are selected from DNA molecules, peptides, proteins, RNA molecules, and transcription factors.
  • the invention provides an isolated cDNA comprising a polynucleotide having the nucleic acid sequence of SEQ ID NO:8 and the complement of SEQ ID NO:8.
  • the cDNA is used as a probe, in an expression vector, and in assays for diagnosis, prognosis, and treatment of disorders associated with atherosclerosis.
  • the invention also provides a composition comprising the cDNA and a labeling moiety.
  • the invention further provides a method for using the cDNA to screen a plurality of molecules to identify a ligand which specifically binds the cDNA, the method comprising combining the cDNA with a sample under conditions to allow specific binding; recovering the bound cDNA; and separating the ligand from the bound cDNA, thereby obtaining purified ligand.
  • the molecules to be screened are selected from DNA molecules, peptides, proteins, RNA molecules, and transcription factors.
  • the invention yet further provides a method for using a cDNA to detect gene expression in a sample containing nucleic acids, the method comprising hybridizing the cDNA to nucleic acids of a sample under conditions for formation of one or more hybridization complexes; and detecting hybridization complex formation, wherein complex formation indicates gene expression in the sample.
  • the cDNA is attached to a substrate.
  • gene expression when compared to standards is diagnostic of a disorder associated with atherosclerosis.
  • the invention provides a vector containing the cDNA, and a host cell containing the vector.
  • the invention also provides a method for producing a peptide or protein, the method comprising culturing the host cell under conditions for expression of the peptide or protein; and recovering the peptide or protein so produced from cell culture.
  • the invention provides a purified peptide or protein comprising an amino acid sequence expressed by a cDNA of the invention.
  • the protein comprises the amino acid sequence of SEQ ID NO:26.
  • the invention additionally provides a composition comprising the protein and a pharmaceutical carrier.
  • the invention also provides a method for using a peptide or protein to screen a plurality of molecules to identify at least one ligand which specifically binds the protein.
  • the molecules to be screened are selected from agonists, antagonists, antibodies, DNA molecules, transcription factors, RNA molecules, and small drug molecules or compounds.
  • the invention further provides a method of using a peptide or protein to purify a ligand.
  • the invention provides a method for using the peptide or protein to produce an antibody which specifically binds the protein.
  • a method for preparing polyclonal antibodies comprises immunizing a animal with peptide or protein under conditions to elicit an antibody response, isolating animal antibodies, attaching the peptide or protein to a substrate, contacting the substrate with isolated antibodies under conditions to allow specific binding to the peptide or protein, dissociating the antibodies from the peptide or protein, thereby obtaining purified polyclonal antibodies.
  • a method for preparing monoclonal antibodies comprises immunizing a animal with a peptide or protein under conditions to elicit an antibody response, isolating antibody producing cells from the animal, fusing the antibody producing cells with immortalized cells in culture to form monoclonal antibody producing hybridoma cells, culturing the hybridoma cells, and isolating monoclonal antibodies from culture.
  • the invention provides purified antibodies which bind specifically to a peptide or protein.
  • the invention also provides a method for using an antibody to detect expression of a peptide or protein in a sample, the method comprising combining the antibody with a sample under conditions for formation of antibody:peptide or protein complexes, and detecting complex formation, wherein complex formation indicates expression of the peptide or protein in the sample.
  • the amount of complex formation when compared to standards is diagnostic of a disorder of the nervous system.
  • the invention provides a method for immunopurification of a protein comprising attaching an antibody to a substrate, exposing the antibody to a sample containing protein under conditions to allow antibody:protein complexes to form, dissociating the protein from the complex, and collecting purified protein.
  • the invention also provides an array upon which a cDNA encoding a protein, the protein, or an antibody which specifically binds the protein are immobilized.
  • the invention also provides a composition comprising a cDNA, a protein, an antibody, or a ligand which has agonistic or antagonistic activity.
  • the invention provides an antibody comprising an antigen binding site, wherein the antigen binding site specifically binds to the protein
  • the invention also provides a method for treating a disorder associated with the differential expression of a cDNA that is coexpressed with one or more known atherosclerosis-associated genes in a subject in need, the method comprising the step of administering to the subject in need the antibody in an amount effective for treating the disorder.
  • the invention further provides an immunoconjugate comprising the antigen binding site of the antibody or joined to a therapeutic agent.
  • the invention additionally provides a method for treating a disorder associated with the differential expression of a cDNA that is coexpressed with one or more known atherosclerosis-associated genes in a subject in need, the method comprising the step of administering to the subject in need the immunoconjugate in an amount effective for treating the disorder.
  • Sequence Listing provides exemplary cDNAs associated with atherosclerosis including polynucleotide sequences SEQ ID NOs:1-25 and the polypeptide sequence, SEQ ID NO:26. Each sequence is identified by a sequence identification number (SEQ ID NO).
  • FIG. 1 shows the cDNA having the nucleic acid sequence of SEQ ID NO:8 which encodes the protein having the amino acid sequence of SEQ ID NO:26.
  • the alignment was produced using MAcDNASIS,PRO software (Hitachi Software Engineering, South San Francisco Calif.).
  • Table 4 shows the-co-expression values (-log p) between the known atherosclerosis-associated genes (abbreviations as shown in Table 3) and the cDNAs of the invention (SEQ IDs).
  • Table 5 summarizes the highly significantly coexpression between each cDNA (SEQ ID) and two known atherosclerosis-associated genes (Gene 1 and Gene 2). P-value and the function or importance of the known atherosclerosis-associated gene are derived from Tables 3 and 4.
  • Table 6 shows transcript images for several of the cDNAs (SEQ ID) of the invention.
  • Column 1 shows the SEQ ID;
  • column 2 the library name;
  • column 3 the number of cDNAs in the library;
  • column 4 the description of the sample from which the library was constructed;
  • column 5 transcript abundance;
  • column 6 percent transcript abundance.
  • Table 7 shows microarray data for several of the cDNAs (SEQ ID) of the invention.
  • Column 1 shows the SEQ ID;
  • column 2 the name of the microarray (GEM) used for the experiment;
  • column 3, the log2 (C5/Cy3 ratio);
  • column 4 the description of the Cy3 sample;
  • column 5 the description of the Cy5 sample.
  • Antibody refers to intact immunoglobulin molecule, a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a recombinant antibody, a humanized antibody, single chain antibodies, a Fab fragment, an F(ab′) 2 fragment, an Fv fragment; and an antibody-peptide fusion protein.
  • Antigenic determinant refers to an antigenic or immunogenic epitope, structural feature, or region of an oligopeptide, peptide, or protein which is capable of inducing formation of an antibody which specifically binds the protein. Biological activity is not a prerequisite for immunogenicity.
  • Array refers to an ordered arrangement of at least two cDNAs, proteins, or antibodies on a substrate. At least one of the cDNAs, proteins, or antibodies represents a control or standard, and the other represents a cDNA, protein, or antibody of diagnostic or therapeutic interest. The arrangement of at least two and up to about 40,000 cDNAs, proteins, or antibodies on the substrate assures that the size and signal intensity of each labeled complex, formed between each cDNA and at least one nucleic acid, each protein and at least one ligand or antibody, or each antibody and at least one protein to which the antibody specifically binds, is individually distinguishable.
  • Atherosclerosis-associated cDNA refers to isolated polynucleotide that exhibits a statistically significant co-expression pattern with known atherosclerosis-associated genes.
  • the cDNAs are represented by SEQ ID NOs:1-25 of the Sequence Listing and the complements of SEQ ID NOs:1-25. They may be of recombinant or synthetic origin, used in its double-stranded or single-stranded form, and combined with vitamins, minerals, carbohydrates, lipids, proteins, other nucleic acids, a pharmaceutical carrier or a labeling moiety to perform a particular activity or form a useful composition.
  • An “atherosclerotic sample or tissue” may be taken using needles, catheters, or scapels and include vessels including the aorta, arteries, arterioles, endothelial cells, plaque, and blood.
  • the sample may contain nucleic acids, proteins, antibodies, and the like. Additionally the sample may comprise the soluble fraction of a cell preparation; an aliquot of media in which cells were grown; a chromosome, an organelle, or membrane isolated or extracted from a cell; genomic DNA, RNA, or cDNA in solution or bound to a substrate.
  • a “combination” comprises at least two sequences selected from SEQ ID NOs:1-25 as presented in the Sequence Listing and the complements of SEQ ID NOs:1-25.
  • “Differential expression” refers to an increased or up-regulated or a decreased or down-regulated expression as detected by absence, presence, or at least two-fold change in the amount of transcribed messenger RNA or translated protein in a sample.
  • disorders associated with atherosclerosis include angina pectoris, coronary artery disease, myocardial infarction, hypertension, transient cerebral ischemia, mesenteric ischemia, peripheral vascular disease, renal artery stenosis, and stroke.
  • An “expression profile” is a representation of gene expression in a sample.
  • a nucleic acid expression profile is produced using sequencing, hybridization, or amplification technologies and mRNAs or cDNAs from a sample.
  • a protein expression profile although time delayed, mirrors the nucleic acid expression profile and uses two-dimensional polyacrylamide electrophoresis (2D-PAGE) and mass spectrophotometry (MS) or western analysis, enzyme-linked immunosorbent assays (ELISAs), fluorescence activated cell sorting (FACS), radioimmunoassays (RIAs), or arrays and labeling moieties or antibodies to detect expression in a sample.
  • the nucleic acids, proteins, or antibodies may be used in solution or attached to a substrate, and their detection is based on methods and labeling moieties well known in the art.
  • a “hybridization complex” is formed between a cDNA of the invention and a nucleic acid of a sample when the purines of one molecule hydrogen bond with the pyrimidines of the complementary molecule, e.g., 5′-A-G-T-C-3′ base pairs with its complete complement, 3′-T-C-A-G-5′.
  • the degree of complementarity and the use of nucleotide analogs affect the efficiency and stringency of hybridization reactions.
  • Identity refers to the quantification (usually percentage) of nucleotide or residue matches between at least two sequences aligned using a standardized algorithm such as Smith-Waterman alignment (Smith and Waterman (1981) J Mol Biol 147:195-197), CLUSTALW (Thompson et al. (1994) Nucleic Acids Res 22:4673-4680), or BLAST2 (Altschul et al. (1997) Nucleic Acids Res 25:3389-340). BLAST2 may be used in a standardized and reproducible way to insert gaps in one of the sequences in order to optimize alignment and to achieve a more meaningful comparison between them.
  • isolated or purified refers to a cDNA or protein that is removed from its natural environment and that is separated from other components with which it is naturally present.
  • Genes known to be associated with atherosclerosis include human 22 kDa smooth muscle protein, calponin (CNN1), pro alpha 1 (I) collagen (COLIA1), collagen alpha-2 type I (COLIA2), collagen alpha-6 type I (COL6A1), procollagen alpha 2(V) (COL5A2), collagen VI alpha-2 (COL6A2), type VI collagen alpha 3 (COL6A3), pro-alpha-i type 3 collagen (COL3A1), pro-alpha-1(V) collagen (COLSA1), matrix Gla protein (MGP), cathepsin K (CSTK), fibrinogen beta chain gene (FBG), pre-pro-von Willebrand factor (VWF), platelet endothelial cell adhesion molecule (PECAM-1), antithrombin III variant (AT3), lipoprotein lipase (LPL), alpha-2-macroglobulin (A2M), apolipoprotein AI (APOA1), apolipoprotein AI (APOA1)
  • Labeleling moiety refers to any reporter molecule whether a visible or radioactive label, stain or dye that can be attached to or incorporated into a cDNA or protein.
  • Visible labels and dyes include but are not limited to anthocyanins, B glucuronidase, BIODIPY, Coomassie blue, Cy3 and Cy5, digoxigenin, FITC, green fluorescent protein, luciferase, spyro red, silver, and the like.
  • Radioactive markers include radioactive forms of hydrogen, iodine, phosphorous, sulfur, and the like.
  • Ligand refers to any agent, molecule, or compound which will bind specifically to a complementary site on a cDNA molecule, a polynucleotide, an epitope or a protein. Such ligands stabilize or modulate the activity of polynucleotides or proteins and may be composed of inorganic or organic substances including nucleic acids, proteins, carbohydrates, fats, and lipids.
  • Markers for disorders associated with atherosclerosis refers to cDNAs, peptides or proteins, and antibodies which are useful in the diagnosis, prognosis, treatment, selection or evaluation of therapies for disorders associated with atherosclerosis. These markers are differentially expressed in samples from subjects predisposed to or manifesting one of these disorders.
  • the known atherosclerosis-associated genes and their contribution and/or function to disorders associated with atherosclerosis are listed in TABLE3.
  • Probe refers to a cDNA of the invention that hybridizes to at least one nucleic acid in a sample. Where targets are single stranded, probes are complementary single strands. Probes can be labeled for use in hybridization reactions including Southern, northern, in situ, dot blot, array, and like technologies or in screening assays.
  • Protein refers to a polypeptide or any portion thereof.
  • An “oligopeptide” is an amino acid sequence from about five residues to about 15 residues that is used as part of a fusion protein to produce an antibody that specifically binds the protein.
  • Specific binding refers to a special and precise interaction between two molecules which is dependent upon their structure, particularly their molecular side groups. For example, the intercalation of a regulatory protein into the major groove of a DNA molecule, the hydrogen bonding along the backbone between two single stranded nucleic acids, or the binding between an epitope of a protein and an agonist, antagonist, or antibody.
  • Substrate refers to any rigid or semi-rigid support to which cDNAs or proteins are bound and includes membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, capillaries or other tubing, plates, polymers, and microparticles with a variety of surface forms including wells, trenches, pins, channels and pores.
  • a “transcript image” is a profile of gene transcription activity in a particular tissue at a particular time. TI provides assessment of the relative abundance of expressed transcripts in the cDNA libraries of an EST database as described in U.S. Pat. No. 5,840,484, incorporated herein by reference.
  • “Variant” refers to molecules that are recognized variations of a polynucleotide or a protein. Splice variants may be determined by BLAST score, wherein the score is at least 100, and most preferably at least 400. Allelic variants have a high percent identity to a polynucleotide and may differ by about three bases per hundred bases. “Single nucleotide polymorphism” (SNP) refers to a change in a single base as a result of a substitution, insertion or deletion. The change may be conservative (purine for purine) or non-conservative (purine to pyrimidine) and may or may not result in a change in an encoded amino acid.
  • SNP single nucleotide polymorphism
  • the present invention encompasses a method for identifying cDNAs that are significantly co-expressed with known atherosclerosis-associated genes.
  • the method identifies a combination of cDNAs useful in diagnosis, prognosis, treatment, and evaluation of therapies for disorders associated with atherosclerosis.
  • the method involves identifying cDNAs that are expressed in a plurality of cDNA libraries. These cDNAs include genes of known or unknown function whose expression patterns are compared with genes having a known function and disease association to determine whether a specified coexpression probability threshold is met. Through this comparison, a subset of the cDNAs having a high coexpression probability with the known genes can be identified.
  • the cDNAs may originate from cDNA libraries derived from a variety of sources including, but not limited to, eukaryotes such as human, mouse, rat, dog, monkey, plant, and yeast; prokaryotes such as bacteria; and viruses.
  • the cDNAs can also be selected from a variety of sequence types including, but not limited to, expressed sequence tags (ESTs), assembled polynucleotide sequences, full length gene coding regions, promoters, introns, enhancers, 5′ untranslated regions, and 3′ untranslated regions.
  • ESTs expressed sequence tags
  • the cDNAs need to be expressed in at least three cDNA libraries.
  • the cDNA libraries used in the coexpression analysis of the present invention can be obtained from adrenal gland, biliary tract, bladder, blood cells, blood vessels, bone marrow, brain, bronchus, cartilage, chromaffin system, colon, connective tissue, cultured cells, embryonic stem cells, endocrine glands, epithelium, esophagus, fetus, ganglia, heart, hypothalamus, immune system, intestine, islets of Langerhans, kidney, larynx, liver, lung, lymph, muscles, neurons, ovary, pancreas, penis, peripheral nervous system, phagocytes, pituitary, placenta, pleurus, prostate, salivary glands, seminal vesicles, skeleton, spleen, stomach, testis, thymus, tongue, ureter, uterus, and the like.
  • the number of cDNA libraries selected can range from as few as 3 to greater than 10,000.
  • the cDNAs are assembled from related sequences, such as assembled sequence fragments derived from a single transcript. Assembly of the sequences can be performed using sequences of various types including, but not limited to, ESTs, extensions, or shotgun sequences. In a most preferred embodiment, the cDNAs are derived from human sequences that have been assembled using the algorithm disclosed in U.S. Ser. No. 09/276,534, filed Mar. 25, 1999, incorporated herein by reference.
  • differential expression of the cDNAs can be evaluated by methods including, but not limited to, differential display by spatial immobilization or by gel electrophoresis, genome mismatch scanning, representational difference analysis, and transcript imaging. Additionally, differential expression can be assessed by microarray technology. These methods may be used alone or in combination.
  • the known atherosclerosis-associated genes were selected based on their function in pathways associated with atherogenesis, their use as diagnostic or prognostic markers, their behavior in model systems or their use as therapeutic targets.
  • the procedure for identifying a cDNA that exhibits a statistically significant coexpression pattern with known atherosclerosis-associated genes is as follows. First, the presence or absence of a gene in a cDNA library is defined: a gene is present in a cDNA library when at least one cDNA fragment corresponding to that gene is detected in a cDNA sample taken from the library, and a gene is absent from a library when no corresponding cDNA fragment is detected in the sample.
  • the significance of gene coexpression is evaluated using a probability method to measure a due-to-chance probability of the coexpression.
  • the probability method can be the Fisher exact test, the chi-squared test, or the kappa test. These tests and examples of their applications are well known in the art and can be found in standard statistics texts (Agresti (1990) Categorical Data Analysis , John Wiley & Sons, New York N.Y.; Rice (1988) Mathematical Statistics and Data Analysis , Duxbury Press, Pacific Grove Calif.).
  • a Bonferroni correction (Rice, supra, p. 384) can also be applied in combination with one of the probability methods for correcting statistical results of one gene vetsus multiple other genes.
  • the due-to-chance probability is measured by a Fisher exact test, and the threshold of the due-to-chance probability is set preferably to less than 0.001, more preferably to less than 0.00001.
  • occurrence data vectors can be generated as illustrated in Table 1. The presence of a gene occurring at least once in a library is indicated by a one, and its absence from the library, by a zero. TABLE 1 Occurrence data for genes A and B Library 1 Library 2 Library 3 . . . Library N gene A 1 1 0 . . . 0 gene B 1 0 1 . . . 0
  • Table 2 presents co-occurrence data for gene A and gene B in a total of 30 libraries. Both gene A and gene B occur 10 times in the libraries. Table 2 summarizes and presents: 1) the number of times gene A and B are both present in a library; 2) the number of times gene A and B are both absent in a library; 3) the number of times gene A is present, and gene B is absent; and 4) the number of times gene B is present, and gene A is absent.
  • the upper left entry is the number of times the two genes co-occur in a library, and the middle right entry is the number of times neither gene occurs in a library.
  • the off diagonal entries are the number of times one gene occurs, and the other does not. Both A and B are present eight times and absent 18 times.
  • Gene A is present, and gene B is absent, two times; and gene B is present, and gene A is absent, two times.
  • the probability (“p-value”) that the above association occurs due to chance as calculated using a Fisher exact test is 0.0003. Associations are generally considered significant if a p-value is less than 0.01 (Agresti, supra; Rice, supra).
  • This method of estimating the probability for co-expression of two genes makes several assumptions. The method assumes that the libraries are independent and are identically sampled. However, in practical situations, the selected cDNA libraries are not entirely independent, because more than one library may be obtained from a single subject or tissue. Nor are they entirely identically sampled, because different numbers of cDNAs may be sequenced from each library. The number of cDNAs sequenced typically ranges from 5,000 to 10,000 cDNAs per library. In addition, because a Fisher exact co-expression probability is calculated for each gene versus 45,233 other assembled genes, a Bonferroni correction for multiple statistical tests is used.
  • the present invention identifies 25 atherosclerosis-associated cDNAs that exhibit strong association with genes known to be specifically expressed in atherosclerosis.
  • Tables 4 and 5 show that the expression of the 25 novel atherosclerosis-associated cDNAs have direct association with the expression of known atherosclerosis-associated genes as described in Table 3 and in the background of the invention. Therefore, the novel atherosclerosis-associated cDNAs can potentially be used in diagnosis, prognosis, treatment or evaluation of therapies fro disorders associated with atherosclerosis. Further, the gene products of the 25 novel atherosclerosis-associated cDNAs are either potential therapeutics or targets for the development of therapeutics against disorders associated with atherosclerosis.
  • the present invention encompasses a combination comprising a plurality of cDNAs having the nucleic acid sequences of SEQ ID NOs:1-25 or the complements of SEQ ID NOs:1-25. These 25 cDNAs have been shown by the method of the present invention to have statistically significant co-expression with known atherosclerosis-associated genes and with each other.
  • the invention also encompasses a cDNA comprising a polynucleotide having the nucleic acid sequence of SEQ ID NO:8 and the complement thereof. As shown in FIG. 1, SEQ ID NO:8 encodes the protein of SEQ ID NO:26.
  • the invention further encompasses a protein comprising the polypeptide having the amino acid sequence of SEQ ID NO:26.
  • the protein encoded by SEQ ID NO:8 has 366 amino acids. Motif analyses of SEQ ID NO:26 shows one potential cAMP- and cGMP-dependent protein kinase phosphorylation site at residue S343, two potential casein kinase II phosphorylation sites at residues S179 and T351, and four potential protein kinase C phosphorylation sites at residues T29, S85, T269, and T324. Additionally, SEQ ID NO:26 contains a potential sugar transport protein signature sequence from residues L201 to S217.
  • cDNAs can be prepared by a variety of synthetic or enzymatic methods well known in the art. cDNAs can be synthesized, in whole or in part, using chemical methods well known in the art (Caruthers et al. (1980) Nucleic Acids Symp Ser (7):215-233). Alternatively, cDNAs can be produced enzymatically or recombinantly, by in vitro or in vivo transcription.
  • Nucleotide analogs can be incorporated into cDNAs by methods well known in the art. The only requirement is that the incorporated analog must base pair with native purines or pyrimidines. For example, 2,6-diaminopurine can substitute for adenine and form stronger bonds with thymidine than those between adenine and thymidine. A weaker pair is formed when hypoxanthine is substituted for guanine and base pairs with cytosine. Additionally, cDNAs can include nucleotides that have been derivatized chemically or enzymatically.
  • cDNAs can be synthesized on a substrate. Synthesis on the surface of a substrate may be accomplished using a chemical coupling procedure and a piezoelectric printing apparatus as described by Baldeschweiler et al. (PCT publication WO95/251116). Alternatively, the cDNAs can be synthesized on a substrate surface using a self-addressable electronic device that controls when reagents are added as described by Heller et al. (U.S. Pat. No. 5,605,662). cDNAs can be synthesized directly on a substrate by sequentially dispensing reagents for their synthesis on the substrate surface or by dispensing preformed DNA fragments to the substrate surface.
  • Typical dispensers include a micropipette delivering solution to the substrate with a robotic system to control the position of the micropipette with respect to the substrate. There can be a multiplicity of dispensers so that reagents can be delivered to the reaction regions efficiently.
  • cDNAs can be immobilized on a substrate by covalent means such as by chemical bonding procedures or UV irradiation.
  • a cDNA is bound to a glass surface which has been modified to contain epoxide or aldehyde groups.
  • a cDNA is placed on a polylysine coated surface and UV cross-linked to it as described by Shalon et al. (WO95/35505).
  • a cDNA is actively transported from a solution to a given position on a substrate by electrical means (Heller, supra). cDNAs do not have to be directly bound to the substrate, but rather can be bound to the substrate through a linker group.
  • the linker groups are typically about 6 to 50 atoms long to provide exposure of the attached cDNA.
  • Preferred linker groups include ethylene glycol oligomers, diamines, diacids and the like.
  • Reactive groups on the substrate surface react with a terminal group of the linker to bind the linker to the substrate. The other terminus of the linker is then bound to the cDNA.
  • polynucleotides, plasmids or cells can be arranged on a filter. In the latter case, cells are lysed, proteins and cellular components degraded, and the DNA is coupled to the filter by UV cross-linking.
  • the cDNAs may be used for a variety of purposes.
  • the combination of the invention may be used on an array.
  • the array in turn, can be used in high-throughput methods for detecting a related polynucleotide in a sample, screening a plurality of molecules or compounds to identify a ligand, diagnosing a disorder such as diabetes, or inhibiting or inactivating a therapeutically relevant gene related to the cDNA.
  • the cDNAs of the invention are employed on an array, the cDNAs are arranged in an ordered fashion so that each cDNA is present at a specified location. Because the cDNAs are at specified locations on the substrate, the hybridization patterns and intensities, which together create a unique, can be interpreted in terms of expression levels of particular genes and can be correlated with a particular metabolic process, condition, disorder, disease, stage of disease, or treatment.
  • the cDNAs or fragments or complements thereof may be used in various hybridization technologies.
  • the cDNAs may be labeled using a variety of reporter molecules by either PCR, recombinant, or enzymatic techniques.
  • a commercially available vector containing the cDNA is transcribed in the presence of an appropriate polymerase, such as T7 or SP6 polymerase, and at least one labeled nucleotide.
  • an appropriate polymerase such as T7 or SP6 polymerase
  • kits are available for labeling and cleanup of such cDNAs.
  • Radioactive Amersham Biosciences (APB), Piscataway N.J.), fluorescent (Qiagen-Operon, Alameda Calif.), and chemiluminescent labeling (Promega, Madison Wis.) are well known in the art.
  • a cDNA may represent the complete coding region of an mRNA or be designed or derived from unique regions of the mRNA or genomic molecule, an intron, a 3′ untranslated region, or from a conserved motif.
  • the cDNA is at least 18 contiguous nucleotides in length and is usually single stranded.
  • Such a cDNA may be used under hybridization conditions that allow binding only to an identical sequence, a naturally occurring molecule encoding the same protein, or an allelic variant. Discovery of related human and mammalian sequences may also be accomplished using a pool of degenerate cDNAs and appropriate hybridization conditions.
  • a cDNA for use in Southern or northern hybridizations may be from about 400 to about 6000 nucleotides long. Such cDNAs have high binding specificity in solution-based or substrate-based hybridizations.
  • An oligonucleotide, a fragment of the cDNA may be used to detect a polynucleotide in a sample using PCR.
  • the stringency of hybridization is determined by G+C content of the cDNA, salt concentration, and temperature. In particular, stringency is increased by reducing the concentration of salt or raising the hybridization temperature. In solutions used for some membrane based hybridizations, addition of an organic solvent such as formamide allows the reaction to occur at a lower temperature.
  • Hybridization may be performed with buffers, such as 5 ⁇ saline sodium citrate (SSC) with 1% sodium dodecyl sulfate (SDS) at 60° C., that permit the formation of a hybridization complex between nucleic acid sequences that contain some mismatches. Subsequent washes are performed with buffers such as 0.2 ⁇ SSC with 0.1% SDS at either 45° C.
  • formamide may be added to the hybridization solution to reduce the temperature at which hybridization is performed. Background signals may be reduced by the use of detergents such as Sarkosyl or TRITON X-100 (Sigma-Aldrich, St. Louis Mo.) and a blocking agent such as denatured salmon sperm DNA. Selection of components and conditions for hybridization are well known to those skilled in the art and are reviewed in Ausubel et al. (1997 , Short Protocols in Molecular Biology , John Wiley & Sons, New York N.Y., Units 2.8-2.11, 3.18-3.19 and 4.6-4.9).
  • Dot-blot, slot-blot, low density and high density arrays are prepared and analyzed using methods known in the art.
  • cDNAs from about 18 consecutive nucleotides to about 5000 consecutive nucleotides in length are contemplated by the invention and used in array technologies.
  • the preferred number of cDNAs on an array is at least about 100,000, a more preferred number is at least about 40,000, an even more preferred number is at least about 10,000, and a most preferred number is at least about 600 to about 800.
  • the array may be used to monitor the expression level of large numbers of genes simultaneously and to identify genetic variants, mutations, and SNPs.
  • Such information may be used to determine gene function; to understand the genetic basis of a disorder; to diagnose a disorder; and to develop and monitor the activities of therapeutic agents being used to control or cure a disorder.
  • a cDNA may be used to screen a library or a plurality of molecules or compounds for a ligand which specifically binds the cDNA.
  • Ligands may be DNA molecules, RNA molecules, peptide nucleic acid molecules, peptides, proteins such as transcription factors, promoters, enhancers, repressors, and other proteins that regulate replication, transcription, or translation of the polynucleotide in the biological system.
  • the assay involves combining the cDNA or a fragment thereof with the molecules or compounds under conditions that allow specific binding and detecting the bound cDNA to identify at least one ligand that specifically binds the cDNA.
  • the cDNA may be incubated with a library of isolated and purified molecules or compounds and binding activity determined by methods such as a gel-retardation assay (U.S. Pat. No. 6,010,849) or a reticulocyte lysate transcriptional assay.
  • the cDNA may be incubated with nuclear extracts from biopsied and/or cultured cells and tissues. Specific binding between the cDNA and a molecule or compound in the nuclear extract is initially determined by gel shift assay. Protein binding may be confirmed by raising antibodies against the protein and adding the antibodies to the gel-retardation assay where specific binding will cause a supershift in the assay.
  • the cDNA may be used to purify a molecule or compound using affinity chromatography methods well known in the art.
  • the cDNA is chemically reacted with cyanogen bromide groups on a polymeric resin or gel. Then a sample is passed over and reacts with or binds to the cDNA. The molecule or compound which is bound to the cDNA may be released from the cDNA by increasing the salt concentration of the flow-through medium and collected.
  • the cDNA may be used to purify a ligand from a sample.
  • a method for using a cDNA to purify a ligand would involve combining the cDNA or a fragment thereof with a sample under conditions to allow specific binding, recovering the bound cDNA, and using an appropriate agent to separate the cDNA from the purified ligand.
  • the full length cDNAs or fragments thereof may be used to produce purified proteins using recombinant DNA technologies described herein and taught in Ausubel (supra; Units 16.1-16.62).
  • One of the advantages of producing proteins by these procedures is the ability to obtain highly-enriched sources of the proteins thereby simplifying purification procedures.
  • the proteins may contain amino acid substitutions, deletions or insertions made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. Such substitutions may be conservative in nature when the substituted residue has structural or chemical properties similar to the original residue (e.g., replacement of leucine with isoleucine or valine) or they may be nonconservative when the replacement residue is radically different (e.g., a glycine replaced by a tryptophan).
  • Expression of a particular cDNA may be accomplished by cloning the cDNA into a vector and transforming this vector into a host cell.
  • the cloning vector used for the construction of cDNA libraries in the LIFESEQ databases may also be used for expression.
  • Such vectors usually contain a promoter and a polylinker useful for cloning, priming, and transcription.
  • An exemplary vector may also contain the promoter for ⁇ -galactosidase, an amino-terminal methionine and the subsequent seven amino acid residues of ⁇ -galactosidase.
  • the vector may be transformed into competent E. coli cells.
  • IPTG isopropylthiogalactoside
  • the cDNA may be shuttled into other vectors known to be useful for expression of protein in specific hosts. Oligonucleotides containing cloning sites and fragments of DNA sufficient to hybridize to stretches at both ends of the cDNA may be chemically synthesized by standard methods. These primers may then be used to amplify the desired fragments by PCR. The fragments may be digested with appropriate restriction enzymes under standard conditions and isolated using gel electrophoresis. Alternatively, similar fragments are produced by digestion of the cDNA with appropriate restriction enzymes and filled in with chemically synthesized oligonucleotides. Fragments of the coding sequence from more than one gene may be ligated together and expressed.
  • a chimeric protein may be expressed that includes one or more additional purification-facilitating domains.
  • additional purification-facilitating domains include, but are not limited to, metal-chelating domains that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex, Seattle Wash.).
  • the inclusion of a cleavable-linker sequence such as ENTEROKINASEMAX (Invitrogen, San Diego Calif.) between the protein and the purification domain may also be used to recover the protein.
  • Suitable host cells may include, but are not limited to, mammalian cells such as Chinese Hamster Ovary (CHO) and human 293 cells, insect cells such as Sf9 cells, plant cells such as Nicotiana tabacum , yeast cells such as Saccharomyces cerevisiae , and bacteria such as E. coli .
  • a useful vector may also include an origin of replication and one or two selectable markers to allow selection in bacteria as well as in a transformed eukaryotic host.
  • Vectors for use in eukaryotic host cells may require the addition of 3′ poly(A) tail if the cDNA lacks poly(A).
  • the vector may contain promoters or enhancers that increase gene expression.
  • Many promoters are known and used in the art. Most promoters are host specific and exemplary promoters includes SV40 promoters for CHO cells; T7 promoters for bacterial hosts; viral promoters and enhancers for plant cells; and PGH promoters for yeast.
  • Adenoviral vectors with the rous sarcoma virus enhancer or retroviral vectors with long terminal repeat promoters may be used to drive protein expression in mammalian cell lines. Once homogeneous cultures of recombinant cells are obtained, large quantities of secreted soluble protein may be recovered from the conditioned medium and analyzed using chromatographic methods well known in the art.
  • An alternative method for the production of large amounts of secreted protein involves the transformation of mammalian embryos and the recovery of the recombinant protein from milk produced by transgenic cows, goats, sheep, and the like.
  • proteins or portions thereof may be produced manually, using solid-phase techniques (Stewart et al. (1969) Solid - Phase Peptide Synthesis , W H Freeman, San Francisco Calif.; Merrifield (1963) J Am Chem Soc 5:2149-2154), or using machines such as the 431A peptide synthesizer (Applied Biosystems (ABI), Foster City Calif.). Proteins produced by any of the above methods may be used as pharmaceutical compositions to treat disorders associated with null or inadequate expression of the genomic sequence.
  • a protein or a portion thereof encoded by the cDNA may be used to screen a library or a plurality of molecules or compounds for a ligand with specific binding affinity or to purify a molecule or compound from a sample.
  • the protein or portion thereof employed in such screening may be free in solution, affixed to an abiotic or biotic substrate, or located intracellularly.
  • viable or fixed prokaryotic host cells that are stably transformed with recombinant nucleic acids that have expressed and positioned a protein on their cell surface can be used in screening assays. The cells are screened against a library or a plurality of ligands and the specificity of binding or formation of complexes between the expressed protein and the ligand may be measured.
  • the ligands may be agonists, antagonists, antibodies, DNA molecules, enhancers, small drug molecules, immunoglobulins, inhibitors, mimetics, peptide nucleic acid molecules, peptides, pharmaceutical agents, proteins, and regulatory proteins, repressors, RNA molecules, ribozymes, transcription factors, or any other test molecule or compound that specifically binds the protein.
  • An exemplary assay involves combining the mammalian protein or a portion thereof with the molecules or compounds under conditions that allow specific binding and detecting the bound protein to identify at least one ligand that specifically binds the protein.
  • This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding the protein specifically compete with a test compound capable of binding to the protein or oligopeptide or fragment thereof.
  • a test compound capable of binding to the protein or oligopeptide or fragment thereof.
  • One method for high throughput screening using very small assay volumes and very small amounts of test compound is described in U.S. Pat. No. 5,876,946. Molecules or compounds identified by screening may be used in a model system to evaluate their toxicity, diagnostic, or therapeutic potential.
  • the protein may be used to purify a ligand from a sample.
  • a method for using a protein to purify a ligand would involve combining the protein or a portion thereof with a sample under conditions to allow specific binding, recovering the bound protein, and using an appropriate chaotropic agent to separate the protein from the purified ligand.
  • a protein encoded by a cDNA of the invention may be used to produce specific antibodies.
  • Antibodies may be produced using an oligopeptide or a portion of the protein with inherent immunological activity. Methods for producing antibodies include: 1) injecting an animal, usually goats, rabbits, or mice, with the protein, or an antigenically-effective portion or an oligopeptide thereof, to induce an immune response; 2) engineering hybridomas to produce monoclonal antibodies; 3) inducing in vivo production in the lymphocyte population; or 4) screening libraries of recombinant immunoglobulins. Recombinant immunoglobulins may be produced as taught in U.S. Pat. No. 4,816,567.
  • Antibodies produced using the proteins of the invention are useful for the diagnosis of prepathologic disorders as well as the diagnosis of chronic or acute diseases characterized by abnormalities in the expression, amount, or distribution of the protein.
  • a variety of protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies specific for proteins are well known in the art. Immunoassays typically involve the formation of complexes between a protein and its specific binding molecule or compound and the measurement of complex formation.
  • Immunoassays may employ a two-site, monoclonal-based assay that utilizes monoclonal antibodies reactive to two noninterfering epitopes on a specific protein or a competitive binding assay (Pound (1998) Immunochemical Protocols , Humana Press, Totowa N.J.).
  • Immunoassay procedures may be used to quantify expression of the protein in cell cultures, in subjects with a particular disorder or in model animal systems under various conditions. Increased or decreased production of proteins as monitored by immunoassay may contribute to knowledge of the cellular activities associated with developmental pathways, engineered conditions or diseases, or treatment efficacy.
  • the quantity of a given protein in a given tissue may be determined by performing immunoassays on freeze-thawed detergent extracts of biological samples and comparing the slope of the binding curves to binding curves generated by purified protein.
  • an antibody array can be used to study protein-protein interactions and phosphorylation.
  • a variety of protein ligands are immobilized on a membrane using methods well known in the art. The array is incubated in the presence of cell lysate until protein:antibody complexes are formed. Proteins of interest are identified by exposing the membrane to an antibody specific to the protein of interest.
  • a protein of interest is labeled with digoxigenin (DIG) and exposed to the membrane; then the membrane is exposed to anti-DIG antibody which reveals where the protein of interest forms a complex.
  • DIG digoxigenin
  • the identity of the proteins with which the protein of interest interacts is determined by the position of the protein of interest on the membrane.
  • Antibody arrays can also be used for high-throughput screening of recombinant antibodies. Bacteria containing antibody genes are robotically-picked and gridded at high density (up to 18,342 different double-spotted clones) on a filter. Up to 15 antigens at a time are used to screen for clones to identify those that express binding antibody fragments. These antibody arrays can also be used to identify proteins which are differentially expressed in samples (de Wildt et al. (2000) Nature Biotechnol 18:989-94).
  • reporter molecules and conjugation techniques are known by those skilled in the art and may be used in various cDNA, polynucleotide, protein, peptide or antibody assays. Synthesis of labeled molecules may be achieved using commercial kits for incorporation of a labeled nucleotide such as 32 P-dCTP, Cy3-dCTP or Cy5-dCTP or amino acid such as 35 S-methionine. Polynucleotides, cDNAs, proteins, or antibodies may be directly labeled with a reporter molecule by chemical conjugation to amines, thiols and other groups present in the molecules using reagents such as BIODIPY or FITC (Molecular Probes, Eugene Oreg.).
  • reagents such as BIODIPY or FITC (Molecular Probes, Eugene Oreg.).
  • the proteins and antibodies may be labeled for purposes of assay by joining them, either covalently or noncovalently, with a reporter molecule that provides for a detectable signal.
  • a reporter molecule that provides for a detectable signal.
  • a wide variety of labels and conjugation techniques are known and have been reported in the scientific and patent literature including, but not limited to U.S. Pat. No. 3,817,837; U.S. Pat. No. 3,850,752; U.S. Pat. No. 3,939,350; U.S. Pat. No. 3,996,345; U.S. Pat. No. 4,277,437; U.S. Pat. No. 4,275,149; and U.S. Pat. No. 4,366,241.
  • the cDNAs, or fragments thereof, may be used to detect and quantify differential gene expression; absence, presence, or excess expression of mRNAs; or to monitor mRNA levels during therapeutic intervention.
  • Disorders associated with atherosclerosis include angina pectoris, coronary artery disease, myocardial infarction, hypertension, transient cerebral ischemia, mesenteric ischemia, peripheral vascular disease, renal artery stenosis, and stroke.
  • These cDNAs can also be utilized as markers of treatment efficacy against the disorders noted above and other disorders, conditions, and diseases over a period ranging from several days to months.
  • the diagnostic assay may use hybridization or amplification technology to compare gene expression in a biological sample from a patient to standard samples in order to detect altered gene expression. Qualitative or quantitative methods for this comparison are well known in the art.
  • the cDNA may be labeled by standard methods and added to a biological sample from a patient under conditions for hybridization complex formation. After an incubation period, the sample is washed and the amount of label (or signal) associated with hybridization complexes is quantified and compared with a standard value. If the amount of label in the patient sample is significantly altered in comparison to the standard value, then the presence of the associated condition, disease or disorder is indicated.
  • a normal or standard expression profile is established. This may be accomplished by combining a biological sample taken from normal subjects, either animal or human, with a probe under conditions for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained using normal subjects with values from an experiment in which a known amount of a purified target sequence is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a particular condition, disease, or disorder. Deviation from standard values toward those associated with a particular condition is used to diagnose that condition.
  • Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies and in clinical trial or to monitor the treatment of an individual patient. Once the presence of a condition is established and a treatment protocol is initiated, diagnostic assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in a normal subject. The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
  • a gene expression profile comprises a plurality of cDNAs and a plurality of detectable hybridization complexes, wherein each complex is formed by hybridization of one or more probes to one or more complementary nucleic acids in a sample.
  • the cDNAs of the invention are used as elements on a array to analyze gene expression profiles.
  • the array is used to monitor the progression of disease.
  • researchers or clinicians can catalog the differences in gene expression between healthy and diseased tissues or cells. By analyzing changes in patterns of gene expression, disease can be diagnosed at earlier stages before the patient is symptomatic.
  • the invention can be used to formulate a prognosis and to design a treatment regimen.
  • the invention can also be used to monitor the efficacy of treatment.
  • the array is employed to improve the treatment regimen.
  • a dosage is established that causes a change in genetic expression patterns indicative of successful treatment. Expression patterns associated with the onset of undesirable side effects are avoided. This approach may be more sensitive and rapid than waiting for the patient to show inadequate improvement, or to manifest side effects, before altering the course of treatment.
  • expression profiles can also be evaluated by methods including, but not limited to, differential display by spatial immobilization or by gel electrophoresis, labeling with radionuclide and quantification using a scintillation counter, genome mismatch scanning, representational difference analysis, transcript imaging, quantitative PCR, and by protein or antibody arrays.
  • Expression profiles produced by these methods may be contrasted with expression profiles produced using normal or diseased tissues.
  • Zweiger 2001 , Transducing the Genome . McGraw-Hill, San Francisco, Calif.
  • Glavas et al. 2001; T cell activation upregulates cyclic nucleotide phosphodiesterases 8A1 and 7A3, Proc Natl Acad Sci 98:6319-6342) among others.
  • animal models which mimic a human disease can be used to produce expression profiles associated with a particular condition, disorder or disease; or treatment of the condition, disorder or disease. Novel treatment regimens may be tested in these animal models using arrays to establish and then follow expression profiles over time.
  • arrays may be used with cell cultures or tissues removed from animal models to rapidly screen large numbers of candidate drug molecules, looking for ones that produce an expression profile similar to those of known therapeutic drugs, with the expectation that molecules with the same expression profile will likely have similar therapeutic effects.
  • the invention provides the means to rapidly determine the molecular mode of action of a drug.
  • Antibodies directed against antigenic determinants of a protein encoded by a cDNA of the invention may be used in assays to quantify the amount of protein found in a particular human cell. Such assays include methods utilizing the antibody and a label to detect expression level under normal or disease conditions.
  • the antibodies may be used with or without modification, and labeled by joining them, either covalently or noncovalently, with a labeling moiety.
  • Protocols for detecting and measuring protein expression using either polyclonal or monoclonal antibodies are well known in the art. Examples include, but are not limited to, western analysis, ELISA, RIA, FACS, and arrays. Such immunoassays typically involve the formation of complexes between the protein and its specific antibody and the measurement of such complexes. These assays are specifically described in Pound (supra).
  • cDNAs and fragments thereof can be used in gene therapy.
  • cDNAs can be delivered ex vivo to target cells, such as cells of bone marrow. Once stable integration and transcription and or translation are confirmed, the bone marrow may be reintroduced into the subject. Expression of the protein encoded by the cDNA may correct a disorder associated with mutation of a normal sequence, reduction or loss of an endogenous target protein, or overepression of an endogenous or mutant protein.
  • cDNAs may be delivered in vivo using vectors such as retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, and bacterial plasmids.
  • Non-viral methods of gene delivery include cationic liposomes, polylysine conjugates, artificial viral envelopes, and direct injection of DNA (Anderson (1998) Nature 392:25-30; Dachs et al. (1997) Oncol Res 9:313-325; Chu et al. (1998) J Mol Med 76(3-4):184-192; Weiss et al. (1999) Cell Mol Life Sci 55(3):334-358; Agrawal (1996) Antisense Therapeutics , Humana Press, Totowa N.J.; and August et al. (1997) Gene Therapy ( Advances in Pharmacology , Vol. 40), Academic Press, San Diego Calif.).
  • expression of a particular protein can be regulated through the specific binding of a fragment of a cDNA to a genomic sequence or an mRNA which encodes the protein or directs its transcription or translation.
  • the cDNA can be modified or derivatized to any RNA-like or DNA-like material including peptide nucleic acids, branched nucleic acids, and the like. These sequences can be produced biologically by transforming an appropriate host cell with a vector containing the sequence of interest.
  • Molecules which regulate the activity of the cDNA or encoded protein are useful as therapeutics for diabetes mellitus, obesity, hypertension, atherosclerosis, polycystic ovarian syndrome, and cancers including breast, prostate, and colon.
  • Such molecules include agonists which increase the expression or activity of the polynucleotide or encoded protein, respectively; or antagonists which decrease expression or activity of the polynucleotide or encoded protein, respectively.
  • an antibody which specifically binds the protein may be used directly as an antagonist or indirectly as a delivery mechanism for bringing a pharmaceutical agent to cells or tissues which express the protein.
  • any of the proteins, or their ligands, or complementary nucleic acid sequences may be administered as pharmaceutical compositions or in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles.
  • the combination of therapeutic agents may act synergistically to affect the treatment or prevention of the conditions and disorders associated with an immune response. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
  • the therapeutic agents may be combined with pharmaceutically-acceptable carriers including excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration used by doctors and pharmacists may be found in the latest edition of Remington's Pharmaceutical Sciences (Mack Publishing, Easton Pa.).
  • Animal models may be used as bioassays where they exhibit a phenotypic response similar to that of humans and where exposure conditions are relevant to human exposures. Mammals are the most common models, and most infectious agent, cancer, drug, and toxicity studies are performed on rodents such as rats or mice because of low cost, availability, lifespan, reproductive potential, and abundant reference literature. Inbred and outbred rodent strains provide a convenient model for investigation of the physiological consequences of underexpression or overexpression of genes of interest and for the development of methods for diagnosis and treatment of diseases. A mammal inbred to overexpress a particular gene (for example, secreted in milk) may also serve as a convenient source of the protein expressed by that gene.
  • Transgenic rodents that overexpress or underexpress a gene of interest may be inbred and used to model human diseases or to test therapeutic or toxic agents.
  • the introduced gene may be activated at a specific time in a specific tissue type during fetal or postnatal development. Expression of the transgene is monitored by analysis of phenotype, of tissue-specific mRNA expression, or of serum and tissue protein levels in transgenic animals before, during, and after challenge with experimental drug therapies.
  • Embryonic (ES) stem cells isolated from rodent embryos retain the potential to form embryonic tissues.
  • ES cells such as the mouse 129/SvJ cell line are placed in a blastocyst from the C57BL/6 mouse strain, they resume normal development and contribute to tissues of the live-born animal.
  • ES cells are preferred for use in the creation of experimental knockout and knockin animals.
  • the method for this process is well known in the art and the steps are: the cDNA is introduced into a vector, the vector is transformed into ES cells, transformed cells are identified and microinjected into mouse cell blastocysts, blastocysts are surgically transferred to pseudopregnant dams.
  • the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains.
  • a region of a gene is enzymatically modified to include a non-natural intervening sequence such as the neomycin phosphotransferase gene (neo; Capecchi (1989) Science 244:1288-1292).
  • the modified gene is transformed into cultured ES cells and integrates into the endogenous genome by homologous recombination.
  • the inserted sequence disrupts transcription and translation of the endogenous gene.
  • ES cells can be used to create knockin humanized animals or transgenic animal models of human diseases. With knockin technology, a region of a human gene is injected into animal ES cells, and the human sequence integrates into the animal cell genome. Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on the progression and treatment of the analogous human condition.
  • cDNAs As described herein, the uses of the cDNAs, provided in the Sequence Listing of this application, and their encoded proteins are exemplary of known techniques and are not intended to reflect any limitation on their use in any technique that would be known to the person of average skill in the art.
  • the cDNAs provided in this application may be used in molecular biology techniques that have not yet been developed, provided the new techniques rely on properties of nucleotide sequences that are currently known to the person of ordinary skill in the art, e.g., the triplet genetic code, specific base pair interactions, and the like.
  • reference to a method may include combining more than one method for obtaining or assembling full length cDNA sequences that will be known to those skilled in the art.
  • the cDNA library SMCCNOS01 was selected as an example to demonstrate the construction of cDNA libraries from which the cDNAs co-expressed with known atherosclerosis-associated genes were derived.
  • the SMCCNOS01 subtracted coronary artery smooth muscle cell library was constructed using 7.56 ⁇ 10 6 clones from the SMCCNOT02 library and was subjected to two rounds of subtraction hybridization for 48 hours with 6.12 ⁇ 10 6 clones from SMCCNOT01.
  • the SMCCNOT02 library was constructed using RNA isolated from coronary artery smooth muscle cells removed from a 3-year-old Caucasian male. The cells were treated for 20 hours with TNF ⁇ and IL-1 ⁇ at 10 ng/ml each.
  • the SMCCNOT01 was constructed using RNA isolated from untreated coronary artery smooth muscle cells from the same donor. Subtractive hybridization conditions were based on the methodologies of Swaroop et al. (1991; Nucleic Acids Res 19:1954) and Bonaldo et al. (1996; Genome Research 6:791).
  • the mRNA-oligo d(T) 25 bound streptavidin particles were separated from the supernatant, washed twice with hybridization buffer 1 (0.15M NaCl, 0.01M Tris-HCl pH 8.0, 1 mM EDTA, 0.1% lauryl sarcosinate) using magnetic separation at each step to remove the supernatant from the particles.
  • hybridization buffer 1 0.15M NaCl, 0.01M Tris-HCl pH 8.0, 1 mM EDTA, 0.1% lauryl sarcosinate
  • RNA was used according to the recommended protocols in the SUPERSCRIPT plasmid system (Invitrogen).
  • the cDNAs were fractionated on a SEPHAROSE CLIB column (APB), and those cDNAs exceeding 400 bp were ligated into pINCY plasmid (Incyte Genomics, Palo Alto Calif.).
  • Recombinant plasmids were transformed into DH5 ⁇ competent cells or ELECTROMAX cells (Invitrogen).
  • Plasmid DNA was released from the cells and purified using the REAL Prep 96 plasmid kit (Qiagen, Valencia Calif.). The recommended protocol was employed except for the following changes: 1) the bacteria were inoculated into 1 ml of sterile TERRIFIC BROTH (BD Biosciences, San Jose Calif.) with carbenicillin at 25 mg/l and glycerol at 0.4%; 2) the cells were cultured for 19 hours and then lysed with 0.3 ml of lysis buffer; 3) following isopropanol precipitation, the plasmid DNA pellet was resuspended in 0.1 ml distilled water, and 4) the samples were transferred to a 96-well block for storage at 4° C.
  • TERRIFIC BROTH BD Biosciences, San Jose Calif.
  • cDNAs were prepared using a MICROLAB 2200 system (Hamilton, Reno Nev.) in combination with the DNA ENGINE thermal cycler (MJ Research, Watertown Mass.). cDNAs were sequenced by the method of Sanger et al. (1975, J Mol Biol 94:441-446) using PRISM 377 (ABI) or MEGABACE 1000 sequencing systems (APB).
  • sequences used for co-expression analysis were assembled from EST sequences, 5′ and 3′ longread sequences, and full length coding sequences.
  • the cDNAs claimed herein were expressed in at least three libraries.
  • the assembly process is described as follows. EST sequence chromatograms were processed and verified. Quality scores were obtained using PHRED (Ewing et al. (1998) Genome Res 8:175-185; Ewing and Green (1998) Genome Res 8:186-194), and edited sequences were loaded into a relational database management system (RDBMS). The sequences were clustered using BLAST with a product score of 50. All clusters of two or more sequences created a bin which represents one transcribed gene.
  • RDBMS relational database management system
  • Bins were annotated by screening the consensus sequence in each bin against public databases, such as GBpri and GenPept from NCBI.
  • the annotation process involved a FASTn screen against the GBpri database in GenBank. Those hits with a percent identity of greater than or equal to 75% and an alignment length of greater than or equal to 100 base pairs were recorded as homolog hits.
  • the residual unannotated sequences were screened by FASTx against GenPept. Those alignments with an E value of less than or equal to 10 ⁇ 8 were recorded as homologs.
  • Sequences were then reclustered using BLASTn and Cross-Match, a program for rapid amino acid and nucleic acid sequence comparison and database search (Green, supra), sequentially. Any BLAST alignment between a sequence and a consensus sequence with a score greater than 150 was realigned using cross-match. The sequence was added to the bin whose consensus sequence gave the highest Smith-Waterman score (Smith et al. (1992) Protein Engineering 5:35-51) amongst local alignments with at least 82% identity. Non-matching sequences were moved into new bins, and assembly processes were repeated.
  • Known atherosclerosis-associated genes were selected to identify the cDNAs that are closely associated with atherosclerosis.
  • the known atherosclerosis-associated genes which were used in this analysis all occur within the LIFESEQ Gold database (Incyte Genomics), and brief descriptions of their functions as they have been reported in the literature are listed in Table 3.
  • Table 3 TABLE 3 Descriptions of Known Atherosclerosis-Associated Genes GENE DESCRIPTION AND REFERENCES Human 22kDa Smooth muscle cell-specific gene which is down-regulated during smooth smooth muscle muscle cell dedifferentiation as part of atherogenic process (Sobue et al. (1998) protein (SM22) Horm Res 50(S2):15-24; Sobue et al.
  • Calponin is smooth muscle-specific and may mediate smooth muscle contractility through binding of the amino-terminal end of the myosin regulatory light chain. Involved in phenotypic modulation of smooth muscle cells, a feature of atherosclerosis (Szymanski et al.(1999) Biochemistry 38:3778-84) pro alpha 1(I) Member of family of fibrous structural proteins. Most abundant structural collagen (COL1A1) component of the extracellular matrix. Secreted as procollagen and converted to collagen by matrix metalloproteinases.
  • Collagens are important in atherosclerosis for promoting platelet aggregation and for providing sites for platelet adhesion to the vessel wall (Wen et al. (1999) Arterioscler Thromb Vasc Biol 19:519-24) collagen alpha-2 type see COL1A1 above I (COL1A2) COL6A1 see COL1A1 above procollagen alpha see COL1A1 above 2(V) (COL5A2) collagen VI alpha-2 see COL1A1 above (COL6A2) type VI collagen see COL1A1 above alpha3 (COL6A3) pro-alpha-1 type 3 see COL1A1 above collagen (COL3A1) pro-alpha-1 (V) see COL1A1 above collagen (COL3A1) matrix Gla protein Role in active calcification of vascular smooth muscle cells, suggested by (MGP) expression study on VSMC in vitro differentiation study.
  • MGP matrix Gla protein Role in active calcification of vascular smooth muscle cells
  • Fibrin deposition is an integral gene (FGB) part of advanced atherosclerotic lesion development. Variation at the beta fibrinogen locus associated with peripheral atherosclerosis (Sueishi et al. (1998) Semin Thromb Hemost 24:255-260; Fowkes et al.(1992) Lancet 339:693-696) pre-pro-von Blood glycoprotein involved in normal hemostasis. Mediates adhesion of Willebrand factor platelets to sites of vascular damage. Also acts as a cofactor in factor VIII (VWF) activity in blood coagulation. Increased levels of VWF are found in atherosclerosis and in several of its major risk factors, including hypercholesterolemia, diabetes, obesity, hypertension.
  • VWF factor VIII
  • LPL lipoprotein lipase Hydrolyses triglyceride in chylomicrons and therefore regulates metabolism of (LPL) circulating lipoproteins.
  • Increased activity of LPL is found in atherosclerotic arteries when compared to normal. Expressed by macrophages in atherosclerotic lesions. Mutations in LPL responsible for familial hypercholesterolemia and premature atherosclerosis (Fisher et al. (1997) Atherosclerosis 135:145-159; Goldberg (1996) J Lipid Res 37:693-707; Gerdes et al.
  • Transgenic mice expressing varying levels of APOAII show increased atherosclerotic lesions than wt when fed an atherogenic diet.
  • Possible interaction between diet/genotype and atherogenic potential Escola-Gil et al. (1998) J Lipid Res 39:457-462; Warden et al. (1993) Proc Natl Acad Sci 90:10886-10890) apolipoprotein B-100 Main apolipoprotein of chylomicrons and low density lipoproteins.
  • Mutations in (APOB) underly familial defective apolipoprotein B-100 in which patients suffer from premature atherosclerosis. Mutations result in defect in binding of LDL to LDL receptor, and accumulation of plasma LDL.
  • SstI RFLP in apoCIII is associated with plasma triglyceride and apoCIII levels and hyperlipidemic phenotypes (Henderson et al.(1987) Hum Genet 75:62-65) apolipoprotein apoC- APOC4 is a lipid-binding protein that has the potential to alter lipid metabolism.
  • IV APOC4
  • Human APOC4 transgenic mice are hypertriglyceridaemic compared to normal controls (Allan et al. (1996) J Lipid Res 37:1510-1518) macrophage Mediates binding, internalisation and processing of negatively-charged macro- scavenger receptor molecules.
  • perilipin Lipid storage droplets of steroidogenic cells are surrounded by perilipins, family of phosphorylated proteins encoded by a singlegene, detected in adipocytes and steroidogenic cells.
  • Possible role in lipid metabolism (Brasaemle et al. (1997) J Biol Chem 272:9378-9387)
  • Prostaglandin D2 Catalyses conversion of PGH2 to PGD2, a prostaglandin important in smooth synthase (PTGDS) muscle contraction/relaxation and potent inhibitor of platelet aggregation.
  • Northern analysis shows strong specific expression in heart.
  • ANX2 is an important anti-inflammatory molecule that binds plasminogen and t- PA and is suspected of having a role in atherogenesis.
  • ANXI is an important anti-inflammatory molecule (Wallner et al. (1986) Nature 320:77-81) Secreted protein, Extracellular glycoprotein secreted by endothelial cells which has a suspected acidic and rich in role in calcification of atherosclerotic plaques.
  • SPARC PDGF-B cysteine
  • cDNAs comprising the polynucleotides of SEQ ID NOs:1-25 and their complements, were identified by their highly significant co-expression with known atherosclerosis-associated genes.
  • Transcript images were performed for several of the cDNAs of the invention using the LIFESEQ GOLD database (July 02 release, Incyte Genomics). This process allowed assessment of the relative abundance of the expressed polynucleotides in all of the cDNA libraries, but those in the cardiovascular category are specifically emphasized. Criteria for transcript imaging can be selected from category, number of cDNAs per library, library description, disease indication, clinical relevance of sample, and the like.
  • All sequences and cDNA libraries in the LIFESEQ database have been categorized by system, organ/tissue and cell type. For each category, the number of libraries in which the sequence was expressed were counted and shown over the total number of libraries in that category. For each library, the number of cDNAs were counted and shown over the total number of cDNAs in that library.
  • all normalized or subtracted libraries which have high copy number sequences removed prior to processing, and all mixed or pooled tissues, which are considered non-specific in that they contain more than one tissue type or more than one subject's tissue, can be excluded from the analysis. Treated and untreated cell lines and/or fetal tissue data can also be excluded where clinical relevance is emphasized.
  • fetal tissue can be emphasized wherever elucidation of inherited disorders or differentiation of particular adult or embryonic stem cells into tissues or organs such as heart, kidney, nerves or pancreas would be aided by removing clinical samples from the analysis.
  • Transcript imaging can also be used to support data from other methodologies such as guilt-by-association and hybridization analyses.
  • the transcript images for SEQ ID NOs:3-6, 8,13, 15-20, and 22 are shown in Table 6.
  • the first column shows library name; the second column, the number of cDNAs sequenced in that library; the third column, the description of the library; the fourth column, absolute abundance of the transcript in the library; and the fifth column, percentage abundance of the transcript in the library.
  • the normal library shows differential expression of the cDNA; in others, the induced or diseased library shows differential expression.
  • the endothelial cells treated with growth factors show 2-8 ⁇ higher expression than untreated endothelial cells in the same experiment.
  • the polypeptide sequence was also analyzed for known motif patterns using MOTIFS, SPSCAN, BLIMPS, and HMM-based protocols.
  • MOTIFS Genetics Computer Group, Madison Wis.
  • SPSCAN Genetics Computer Group searches polypeptide sequences for patterns that match those defined in the Prosite Dictionary of Protein Sites and Patterns (Bairoch, supra) and displays the patterns found and their corresponding literature abstracts.
  • SPSCAN Genetics Computer Group searches for potential signal peptide sequences using a weighted matrix method (Nielsen et al. (1997) Protein Engineering 10:1-6). Hits with a score of 5 or greater were considered.
  • BLIMPS uses a weighted matrix analysis algorithm to search for sequence similarity between the polypeptide sequences and those contained in BLOCKS, a database consisting of short amino acid segments, or blocks of 3-60 amino acids in length, compiled from the PROSITE database (Henikoff; supra; Bairoch, supra), and those in PRINTS, a protein fingerprint database based on non-redundant sequences obtained from sources such as SwissProt, GenBank, PIR, and NRL-3D (Attwood et al. (1997) J Chem Inf Comput Sci 37:417-424).
  • the BLIMPS searches reported matches with a cutoff score of 1000 or greater and a cutoff probability value of 1.0 ⁇ 10 ⁇ 3 .
  • HMM-based protocols were based on a probabilistic approach and searched for consensus primary structures of gene families in the protein sequences (Eddy, supra; Sonnhammer, supra). More than 500 known protein families with cutoff scores ranging from 10 to 50 bits were selected for use in this invention.
  • SEQ ID NO:1-25 are represented among the template sequences in the LIFESEQ GOLD database (Incyte Genomics). Several of these sequences, specifically SEQ ID NOs:1, 2, 10, 12, 18, and 32-34 have been used on microarrays in experiments investigating differential gene expression in cardiovascular samples. Table 7 presents the results of these experiments; results were significant if the log2 Cy/Cy5 ratio exceeded ⁇ 1.00 in either the normal or the induced or disease state.
  • Activation of the vascular endothelium is considered to be a central event in a wide range of both physiological and pathophysiological processes, such as vascular tone regulation, coagulation and thrombosis, atherosclerosis, and inflammation.
  • HAEC human aortic endothelial cells
  • HAECs are primary cells derived from the endothelium of a human aorta. They have been used as an experimental model for investigating the role of the endothelium in human vascular biology in vitro. HAECs were grown to 85% confluency, split into two samples, one of which was then treated with growth factors, LDL, cytokines, O2, and the like for variable time periods.
  • ECV304, HUAEC, and HUVEC are endothelial cell lines derived from the endothelium of the human umbilical artery or vein. This cell model has been extensively used to study the functional biology of human endothelial cells. These cells were also grown to about 85% confluency, split into samples, one of which was then treated with growth factors, LDL, cytokines, O2, and the like for variable time periods.
  • TNF- ⁇ is a pleiotropic cytokine that is known to play a central role in the mediation of inflammatory responses through activation of multiple signal transduction pathways.
  • TNF- ⁇ is produced by activated lymphocytes, macrophages, and other white blood cells, and is known to activate endothelial cells. Monitoring the endothelial cells' response to TNF- ⁇ at the level of the mRNA expression can provide information necessary for better understanding of both TNF- ⁇ signaling pathways and endothelial cell biology.
  • PMA is a broad activator of the protein kinase C-dependent pathways.
  • lonomycin is a calcium ionophore that permits the entry of calcium in the cell, hence increasing the cytosolic calcium concentration.
  • the combination of PMA and ionomycin activates two of the major signaling pathways used by mammalian cells to interact with their environment. In T cells, the combination of PMA and ionomycin mimics the type of secondary signaling events elicited during optimal B cell activation.
  • the HUMAN GENOME GEM series 1-5 microarrays (Incyte Genomics) contain 45,320 array elements which represent 22,632 annotated clusters and 22,688 unannotated clusters.
  • Incyte clones were mapped to non-redundant Unigene clusters (Unigene database (build 46), NCBI; Shuler (1997) J Mol Med 75:694-698), and the 5′ clone with the strongest BLAST alignment (at least 90% identity and 100 bp overlap) was chosen, verified, and used in the construction of the microarray.
  • the UNIGEM V 2.0 microarray (Incyte Genomics) contains 8,502 array elements which represent 8,372 annotated genes and 130 unannotated clusters.
  • cDNAs were amplified from bacterial cells using primers complementary to vector sequences flanking the cDNA insert. Thirty cycles of PCR increased the initial quantity of cDNAs from 1-2 ng to a final quantity greater than 5 ⁇ g. Amplified cDNAs were then purified using SEPHACRYL-400 columns (APB). Purified cDNAs were immobilized on polymer-coated glass slides. Glass microscope slides (Corning, Corning N.Y.) were cleaned by ultrasound in 0.1% SDS and acetone, with extensive distilled water washes between and after treatments.
  • Microarrays were UV-crosslinked using a STRATALINKER UV-crosslinker (Stratagene), and then washed at room temperature once in 0.2% SDS and three times in distilled water. Non-specific binding sites were blocked by incubation of microarrays in 0.2% casein in phosphate buffered saline (Tropix, Bedford Mass.) for 30 minutes at 60° C. followed by washes in 0.2% SDS and distilled water as before.
  • STRATALINKER UV-crosslinker Stratagene
  • Cells were harvested and lysed in 1 ml of TRIZOL reagent (5 ⁇ 10 6 cells/ml; Invitrogen). The lysates were vortexed thoroughly and incubated at room temperature for 2-3 minutes and extracted with 0.5 ml chloroform. The extract was mixed, incubated at room temperature for 5 minutes, and centrifuged at 16,000 ⁇ g for 15 minutes at 4° C. The aqueous layer was collected, and an equal volume of isopropanol was added. Samples were mixed, incubated at room temperature for 10 minutes, and centrifuged at 16,000 ⁇ g for 20 minutes at 4° C.
  • RNA pellet was washed with 1 ml of 70% ethanol, centrifuged at 16,000 ⁇ g at 4° C., and resuspended in RNAse-free water. The concentration of the RNA was determined by measuring the optical density at 260 nm.
  • Poly(A) RNA was prepared using an OLIGOTEX mRNA kit (Qiagen) with the following modifications: OLIGOTEX beads were washed in tubes rather than spin columns, resuspended in elution buffer, and then loaded onto spin columns to recover the mRNA. To obtain maximum yield, the mRNA was eluted twice.
  • Each poly(A) RNA sample was reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/ ⁇ l oligo-d(T) primer (21mer), 1 ⁇ first strand buffer, 0.03 units/ul RNAse inhibitor, 500 uM dATP, 500 uM dGTP, 500 uM dTTP, 40 uM dCTP, and 40 uM either dCTP-Cy3 or dCTP-Cy5 (APB).
  • the reverse transcription reaction was performed in a 25 ml volume containing 200 ng poly(A) RNA using the GEMBRIGHT kit (Incyte Genomics).
  • control poly(A) RNAs (YCFRO6, YCFR45, YCFR67, YCFR85, YCFR43, YCFR22, YCFR23, YCFR25, YCFR44, YCFR26) were synthesized by in vitro transcription from non-coding yeast genomic DNA (W. Lei, unpublished).
  • control mRNAs (YCFRO6, YCFR45, YCFR67, and YCFR85) at 0.002 ng, 0.02 ng, 0.2 ng, and 2 ng were diluted into reverse transcription reaction at ratios of 1:100,000, 1:10,000, 1:1000, 1:100 (w/w) to sample mRNA, respectively.
  • control mRNAs (YCFR43, YCFR22, YCFR23, YCFR25, YCFR44, YCFR26) were diluted into reverse transcription reaction at ratios of 1:3, 3:1, 1:10, 10:1, 1:25, 25:1 (w/w) to sample mRNA. Reactions were incubated at 37° C. for 2 hr, treated with 2.5 ml of 0.5M sodium hydroxide, and incubated for 20 minutes at 85° C. to the stop the reaction and degrade the RNA.
  • cDNAs were purified using two successive CHROMA SPIN 30 gel filtration spin columns (Clontech). Cy3- and Cy5-labeled reaction samples were combined as described below and ethanol precipitated using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100% ethanol. The cDNAs were then dried to completion using a SpeedVAC system (Savant Instruments, Holbrook N.Y.) and resuspended in 14 ⁇ l 5 ⁇ SSC, 0.2% SDS.
  • SpeedVAC system Savant Instruments, Holbrook N.Y.
  • Hybridization reactions contained 9 ⁇ l of sample mixture containing 0.2 ⁇ g each of Cy3 and Cy5 labeled cDNA synthesis products in 5 ⁇ SSC, 0.2% SDS hybridization buffer. The mixture was heated to 65° C. for 5 minutes and was aliquoted onto the microarray surface and covered with an 1.8 cm 2 coverslip. The microarrays were transferred to a waterproof chamber having a cavity just slightly larger than a microscope slide. The chamber was kept at 100% humidity internally by the addition of 140 ⁇ l of 5 ⁇ SSC in a corner of the chamber. The chamber containing the microarrays was incubated for about 6.5 hours at 60° C. The microarrays were washed for 10 min at 45° C. in low stringency wash buffer (1 ⁇ SSC, 0.1% SDS), three times for 10 minutes each at 45° C. in high stringency wash buffer (0.1 ⁇ SSC), and dried.
  • Reporter-labeled hybridization complexes were detected with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent, Santa Clara Calif.) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of Cy5.
  • the excitation laser light was focused on the microarray using a 20 ⁇ microscope objective (Nikon, Melville N.Y.).
  • the slide containing the microarray was placed on a computer-controlled X-Y stage on the microscope and raster-scanned past the objective.
  • the 1.8 cm ⁇ 1.8 cm microarray used in the present example was scanned with a resolution of 20 micrometers.
  • the mixed gas multiline laser excited the two fluorophores sequentially. Emitted light was split, based on wavelength, into two photomultiplier tube detectors (PMT R1477; Hamamatsu Photonics Systems, Bridgewater N.J.) corresponding to the two fluorophores. Appropriate filters positioned between the microarray and the photomultiplier tubes were used to filter the signals. The emission maxima of the fluorophores used were 565 nm for Cy3 and 650 nm for Cy5. Each microarray was typically scanned twice, one scan per fluorophore using the appropriate filters at the laser source, although the apparatus was capable of recording the spectra from both fluorophores simultaneously.
  • the sensitivity of the scans was calibrated using the signal intensity generated by a cDNA control species. Samples of the calibrating cDNA were separately labeled with the two fluorophores and identical amounts of each were added to the hybridization mixture. A specific location on the microarray contained a complementary DNA sequence, allowing the intensity of the signal at that location to be correlated with a weight ratio of hybridizing species of 1:100,000.
  • the output of the photomultiplier tube was digitized using a 12-bit RTI-835H analog-to-digital (A/D) conversion board (Analog Devices, Norwood, Mass.) installed in an IBM-compatible PC computer.
  • the digitized data were displayed as an image where the signal intensity was mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal).
  • the data was also analyzed quantitatively. Where two different fluorophores were excited and measured simultaneously, the data were first corrected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore's emission spectrum.
  • a grid was superimposed over the fluorescence signal image such that the signal from each spot was centered in each element of the grid.
  • the fluorescence signal within each element was then integrated to obtain a numerical value corresponding to the average intensity of the signal.
  • the software used for signal analysis was the GEMTOOLS gene expression analysis program (Incyte Genomics). Significance was defined as signal to background ratio exceeding 2 ⁇ and area hybridization exceeding 40%.
  • Clones were aligned against the LIFESEQ Gold 5.1 database (Incyte Genomics) and an Incyte template and its sequence variants were chosen for each clone.
  • the template and variant sequences were aligned against the GenBank nucleotide sequence databases using BLASTn (vers. 2.0, NCBI) to acquire annotation.
  • the template and variant sequences were translated into amino acid sequences which were aligned against GenPept and other protein databases using BLASTp (vers. 2.0, NCBI) to acquire annotation and characterization, i.e., structural motifs.
  • Table 3 shows the GenBank annotations (where available) for SEQ ID NOs:1-25 of this invention as produced by BLAST analysis.
  • Percent sequence identity can be determined electronically for two or more amino acid or nucleic acid sequences using the MEGALIGN program, a component of LASERGENE software (DNASTAR). The percent identity between two amino acid sequences is calculated by dividing the length of sequence A, minus the number of gap residues in sequence A, minus the number of gap residues in sequence B, into the sum of the residue matches between sequence A and sequence B, times one hundred. Gaps of low or of no homology between the two amino acid sequences are not included in determining percentage identity.
  • Sequences with conserved protein motifs may be searched using the BLOCKS search program. This program analyses sequence information contained in the Swiss-Prot and PROSITE databases and is useful for determining the classification of uncharacterized proteins translated from genomic or cDNA sequences (Bairoch, supra; Attwood, supra).
  • PROSITE database is a useful source for identifying functional or structural domains that are not detected using motifs due to extreme sequence divergence. Using weight matrices, these domains are calibrated against the SWISS-PROT database to obtain a measure of the chance distribution of the matches.
  • the PRINTS database can be searched using the BLIMPS search program to obtain protein family “fingerprints”.
  • the PRINTS database complements the PROSITE database by exploiting groups of conserved motifs within sequence alignments to build characteristic signatures of different protein families. For both BLOCKS and PRINTS analyses, the cutoff scores for local similarity were:
  • the cDNAs are applied to a membrane substrate by one of the following methods.
  • a mixture of cDNAs is fractionated by gel electrophoresis and transferred to a nylon membrane by capillary transfer.
  • the cDNAs are individually ligated to a vector and inserted into bacterial host cells to form a library.
  • the cDNAs are then arranged on a substrate by one of the following methods.
  • bacterial cells containing individual clones are robotically picked and arranged on a nylon membrane.
  • the membrane is placed on LB agar containing selective agent (carbenicillin, kanamycin, ampicillin, or chloramphenicol depending on the vector used) and incubated at 37° C. for 16 hr.
  • the membrane is removed from the agar and consecutively placed colony side up in 10% SDS, denaturing solution (1.5 M NaCl, 0.5 M NaOH), neutralizing solution (1.5 M NaCl, 1 M Tris, pH 8.0), and twice in 2 ⁇ SSC for 10 min each.
  • the membrane is then UV irradiated in a STRATALINKER UV-crosslinker (Stratagene).
  • cDNAs are amplified from bacterial vectors by thirty cycles of PCR using primers complementary to vector sequences flanking the insert. PCR amplification increases a starting concentration of 1-2 ng nucleic acid to a final quantity greater than 5 ⁇ g.
  • Amplified nucleic acids from about 400 bp to about 5000 bp in length are purified using SEPHACRYL-400 beads (APB). Purified nucleic acids are arranged on a nylon membrane manually or using a dot/slot blotting manifold and suction device and are immobilized by denaturation, neutralization, and UV irradiation as described above.
  • Hybridization probes derived from cDNAs of the Sequence Listing are employed for screening cDNAs, mRNAs, or genomic DNA in membrane-based hybridizations. Probes are prepared by diluting the cDNAs to a concentration of 40-50 ng in 45 IL TE buffer, denaturing by heating to 100° C. for five min and briefly centrifuging. The denatured cDNA is then added to a REDIPRIME tube (APB), gently mixed until blue color is evenly distributed, and briefly centrifuged. Five microliters of [ 32 P]dCTP is added to the tube, and the contents are incubated at 37° C. for 10 min.
  • APB REDIPRIME tube
  • the labeling reaction is stopped by adding 5 ⁇ l of 0.2M EDTA, and probe is purified from unincorporated nucleotides using a PROBEQUANT G-50 microcolumn (APB).
  • the purified probe is heated to 100° C. for five min and then snap cooled for two min on ice.
  • Membranes are pre-hybridized in hybridization solution containing 1% Sarkosyl and Ix high phosphate buffer (0.5 M NaCl, 0.1 M Na 2 HPO 4 , 5 mM EDTA, pH 7) at 55° C. for two hr.
  • the probe diluted in 15 ml fresh hybridization solution, is then added to the membrane.
  • the membrane is hybridized with the probe at 55° C. for 16 hr.
  • the membrane is washed for 15 min at 25° C. in imM Tris (pH 8.0), 1% Sarkosyl, and four times for 15 min each at 25° C. in 1 mM Tris (pH 8.0).
  • XOMAT-AR film Eastman Kodak, Rochester N.Y. is exposed to the membrane overnight at ⁇ 70° C., developed, and examined.
  • cDNA is subcloned into a vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription.
  • promoters include, but are not limited to, the trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element.
  • Recombinant vectors are transformed into bacterial hosts, such as BL21(DE3). Antibiotic resistant bacteria express the protein upon induction with IPTG.
  • Expression in eukaryotic cells is achieved by infecting Spodoptera frugiperda (Sf9) insect cells with recombinant baculovirus, Autographica californica nuclear polyhedrosis virus.
  • the polyhedrin gene of baculovirus is replaced with the cDNA by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of transcription.
  • the protein is synthesized as a fusion protein with glutathione-S-transferase (GST; APB) or a similar alternative such as FLAG.
  • GST glutathione-S-transferase
  • the fusion protein is purified on immobilized glutathione under conditions that maintain protein activity and antigenicity.
  • the GST moiety is proteolytically cleaved from the protein with thrombin.
  • a fusion protein with FLAG, an 8-amino acid peptide is purified using commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak, Rochester N.Y.).
  • a denatured protein from a reverse phase HPLC separation is obtained in quantities up to 75 mg. This denatured protein is used to immunize mice or rabbits following standard protocols. About 100 ⁇ g is used to immunize a mouse, while up to 1 mg is used to immunize a rabbit. The denatured protein is radioiodinated and incubated with murine B-cell hybridomas to screen for monoclonal antibodies. About 20 mg of protein is sufficient for labeling and screening several thousand clones.
  • the amino acid sequence translated from a cDNA of the invention is analyzed using PROTEAN software (DNASTAR) to determine antigenic determinants of the protein.
  • the optimal sequences for immunization are usually at the C-terminus, the N-terminus, and those intervening, hydrophilic regions of the protein that are likely to be exposed to the external environment when the protein is in its natural conformation.
  • oligopeptides about 15 residues in length are synthesized using an 431 peptide synthesizer (ABI) using Fmoc-chemistry and then coupled to keyhole limpet hemocyanin (KLH; Sigma-Aldrich) by reaction with M-maleimidobenzoyl-N-hydroxysuccinimide ester.
  • a cysteine may be introduced at the N-terminus of the peptide to permit coupling to KLH.
  • Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant. The resulting antisera are tested for antipeptide activity by binding the peptide to plastic, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radioiodinated goat anti-rabbit IgG.
  • Hybridomas are prepared and screened using standard techniques. Hybridomas of interest are detected by screening with radioiodinated protein to identify those fusions producing a monoclonal antibody specific for the protein.
  • wells of 96 well plates FAST, Becton-Dickinson, Palo Alto Calif.
  • affinity-purified, specific rabbit-anti-mouse (or suitable anti-species Ig) antibodies at 10 mg/ml.
  • the coated wells are blocked with 1% BSA and washed and exposed to supernatants from hybridomas. After incubation, the wells are exposed to radiolabeled protein at 1 mg/ml. Clones producing antibodies bind a quantity of labeled protein that is detectable above background.
  • Such clones are expanded and subjected to 2 cycles of cloning at 1 cell/3 wells.
  • Cloned hybridomas are injected into pristane-treated mice to produce ascites, and monoclonal antibody is purified from the ascitic fluid by affinity chromatography on protein A (APB).
  • Monoclonal antibodies with affinities of at least 10 8 M ⁇ , preferably 10 9 to 1010 M ⁇ 1 or stronger, are made by procedures well known in the art.
  • Naturally occurring or recombinant protein is purified by immunoaffinity chromatography using antibodies specific for the protein.
  • An immunoaffinity column is constructed by covalently coupling the antibody to CNBr-activated SEPHAROSE resin (APB). Media containing the protein is passed over the immunoaffinity column, and the column is washed using high ionic strength buffers in the presence of detergent to allow preferential absorbance of the protein. After coupling, the protein is eluted from the column using a buffer of pH 2-3 or a high concentration of urea or thiocyanate ion to disrupt antibody/protein binding, and the protein is collected.
  • APB CNBr-activated SEPHAROSE resin
  • the cDNA or fragments thereof and the protein or portions thereof are labeled with 32 P-dCTP, Cy3-dCTP, Cy5-dCTP (APB), or BIODIPY or FITC (Molecular Probes), respectively.
  • Candidate molecules or compounds previously arranged on a substrate are incubated in the presence of labeled nucleic or amino acid. After incubation under conditions for either a cDNA or a protein, the substrate is washed, and any position on the substrate retaining label, which indicates specific binding or complex formation, is assayed. The binding molecule is identified by its arrayed position on the substrate.

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Abstract

The present invention relates to a combination comprising a plurality of cDNAs which are differentially expressed in cardiovascular diseases. The combination and compositions can be used entirely or in part to diagnose, to stage, to treat, or to monitor the progression or treatment of disorders associated with atherosclerosis.

Description

    FIELD OF THE INVENTION
  • The invention relates to a combination of isolated cDNAs that are significantly co-expressed with one or more known atherosclerosis-associated genes. The invention also relates to the use of the combination in the diagnosis, prognosis, treatment, and evaluation of therapies for disorders associated with atherosclerosis. [0001]
    • BACKGROUND OF THE INVENTION
  • Atherosclerosis is a disorder characterized by cellular changes in the arterial intima and the formation of arterial plaques containing intracellular and extracellular deposits of lipids. The thickening of artery walls and the narrowing of the arterial lumen underlies the pathologic condition in most cases of coronary artery disease, aortic aneurysm, peripheral vascular disease, and stroke. A number of metabolic pathways and a cascade of molecular events is involved in the cellular morphogenesis, proliferation, and cellular migration that results in atherogenesis (Libby et al. (1997) Int J Cardiol 62 (S2):23-29). [0002]
  • The artery walls consist of three layers: the intima (innermost), the media, and the adventitia (outermost). The intima consists of a layer of endothelial cells lining the lumen of arteries and arterioles. Endothelial cells form a barrier against the indiscriminate entry of substances from the blood into the artery. Specific transporter proteins expressed by endothelial cells facilitate barrier function. Endothelial cells also secrete a number of substances which help regulate downstream vascular contractility blood coagulation, and other aspects of vascular biology. The medial layer of the arterial wall contains smooth muscle cells in a matrix of collagen and elastic fibers produced by the smooth muscle cells. Contraction and relaxation of the smooth muscle layer allows arteries and arterioles to modulate blood pressure and blood flow. The outermost layer of the arterial wall, the adventitia, is a mixture of collagen bundles, elastic fibers, some smooth muscle cells, fibroblasts and nerve cells. The adventitia provides structural integrity to the blood vessel and acts as a support matrix for the media and intima. [0003]
  • Initiation of an atherosclerotic lesion often occurs following vascular endothelial cell injury often attributable to hypertension, diabetes mellitus, hyperlipidemia, fluctuating shear stress, smoking, or transplant rejection. Nitric oxide and superoxide anions are released and react to form cytodestructive peroxynitrite radicals that cause injury to the endothelium and myocytes of the intima and lead to expression of a variety of molecules that produce local and systemic effects. These effects include the release of mediators of inflammation such as cytokines, complement components, prostaglandins, and downstream transcription factors. Such mediators promote monocyte infiltration of the vascular intima and lead to the upregulation of adhesion molecules which encourages attachment of the monocytes to the damaged endothelial cells. Simultaneously, components of the extracellular matrix including collagens, fibrinogens, and matrix Gla protein are induced and provide sites for monocyte attachment, and annexins, [0004] plasminogen activator inhibitor 1, and nitric oxide synthases are induced to counteract these effects.
  • Monocytes that infiltrate the lesion accumulate modified low density lipoprotein through scavenger receptors such as CD36 and macrophage scavenger receptor type I. The abundance of modified lipids is a factor in atherogenesis and is influenced by modifying enzymes such as lipoprotein lipase, carboxyl ester lipase, serum amyloid P component, LDL-receptor related protein, microsomal triglyceride transfer protein, and serum esterases such as paraoxonase. Lipid metabolism is governed by cholesterol biosynthesis enzymes such as 3-hydroxy-3-methylglutaryl coenzyme A synthase, and products of the apolipoprotein genes. Modified lipid stabilization and accumulation is aided by perilipin and alpha-2-macroglobulin. [0005]
  • As monocytes accumulate in the lesion, they can rupture and release free cholesterol, cytokines, and procoagulants into the surrounding environment. This is the process that leads to plaque development; plaque consists of a mass of lipid-engorged monocytes and a lipid-rich necrotic core covered by a fibrous cap. The gradual progression of plaque growth is punctuated by thrombus formation and leads to clinical symptoms such as unstable angina, myocardial infarction, or stroke. Thrombus formation is initiated by episodic plaque rupture which exposes flowing blood to tissue factors, which induce coagulation, and collagen, which activates platelets. After initiation of the atherosclerotic lesion, enzymes that degrade extracellular matrix components (ECM) such as matrix metalloproteinases and cathepsin K are up-regulated, and inhibitors of ECM are down-regulated. This results in destabilization of the atherosclerotic lesion and subsequent complications including myocardial infarction, angina, and stroke. Further arterial occlusion and infiltration increase with the expression of coagulation factors and down-regulation of their inhibitors, antithrombin III, and lipoprotein-associated coagulation inhibitor. [0006]
  • Smooth muscle cells build up in the arterial media and constitute one of the principal cell types in atherosclerotic and restenotic lesions. They show a high degree of plasticity and are able to shift between a differentiated, contractile phenotype and a less differentiated, synthetic phenotype. This modulation occurs as a response to factors secreted from cells at the site of vascular injury and results in structural reorganization with a loss of myofilaments and the formation of an extensive endoplasmic reticulum and a large Golgi complex. Genes encoding secreted protein, acidic and rich in cysteine (SPARC) and endothelin-1 contribute to these changes. At the same time, the expression of cytoskeletal proteins such as calponin, myosin, desmin, and other gene products in the cells is altered. As a result, the smooth muscle cells lose their contractility and become able to migrate from the media to the intima, to proliferate, and to secrete extracellular matrix components which contribute to arterial intimal thickening. [0007]
  • The initiation and progression of atherosclerotic lesion development requires the interplay of various molecular pathways Many genes that participate in these processes are known, and some of them have been shown to have a direct role in atherosclerosis pathogenesis by animal model experiments, in vitro assays, and epidemiological studies (Krettek et al. (1997) Arterioscler Thromb Vasc Biol 17:2897-2903; Fisher et al. (1997) Atherosclerosis 135:145-159; Shih et al. (1998) Circulation 95:2684-2693; and Bocan et al. (1998) Atherosclerosis 139:21-30). [0008]
  • The present invention satisfies a need in the art by providing a combination comprising a plurality of cDNAs that are useful for diagnosis, prognosis, treatment, and evaluation of therapies for disorders associated with atherosclerosis. [0009]
    • SUMMARY OF THE INVENTION
  • The invention provides a combination of isolated cDNAs that are significantly co-expressed with one or more known atherosclerosis-associated genes. The combination comprises the isolated cDNAs having the nucleic acid sequences of SEQ ID NOs:1-25 and the complements of SEQ ID NOs:1-25. In one embodiment, the combination is placed on a substrate. In another embodiment, the substrate is a microarray. [0010]
  • The invention also provides a method for using the combination to detect gene expression in a sample containing nucleic acids, the method comprising hybridizing the substrate containing the combination to the nucleic acids of the sample under conditions for formation of one or more hybridization complexes and detecting hybridization complex formation, wherein complex formation indicates gene expression in the sample. In one embodiment, the sample is from artery or is obtained during microsurgery to open a blocked artery. In another embodiment, complex formation is compared to standards and is diagnostic of a disorder associated with atherosclerosis. [0011]
  • The invention further provides a method of using a combination to screen a plurality of molecules to identify at least one ligand which specifically binds a cDNA of the combination, the method comprising combining the substrate containing the combination with molecules under conditions to allow specific binding; and detecting specific binding, thereby identifying a ligand which specifically binds at least one cDNA of the combination. In one embodiment, the molecules are selected from DNA molecules, peptides, proteins, RNA molecules, and transcription factors. [0012]
  • The invention provides an isolated cDNA comprising a polynucleotide having the nucleic acid sequence of SEQ ID NO:8 and the complement of SEQ ID NO:8. In different embodiments, the cDNA is used as a probe, in an expression vector, and in assays for diagnosis, prognosis, and treatment of disorders associated with atherosclerosis. The invention also provides a composition comprising the cDNA and a labeling moiety. The invention further provides a method for using the cDNA to screen a plurality of molecules to identify a ligand which specifically binds the cDNA, the method comprising combining the cDNA with a sample under conditions to allow specific binding; recovering the bound cDNA; and separating the ligand from the bound cDNA, thereby obtaining purified ligand. In one embodiment, the molecules to be screened are selected from DNA molecules, peptides, proteins, RNA molecules, and transcription factors. The invention yet further provides a method for using a cDNA to detect gene expression in a sample containing nucleic acids, the method comprising hybridizing the cDNA to nucleic acids of a sample under conditions for formation of one or more hybridization complexes; and detecting hybridization complex formation, wherein complex formation indicates gene expression in the sample. In one embodiment, the cDNA is attached to a substrate. In another embodiment, gene expression when compared to standards is diagnostic of a disorder associated with atherosclerosis. [0013]
  • The invention provides a vector containing the cDNA, and a host cell containing the vector. The invention also provides a method for producing a peptide or protein, the method comprising culturing the host cell under conditions for expression of the peptide or protein; and recovering the peptide or protein so produced from cell culture. [0014]
  • The invention provides a purified peptide or protein comprising an amino acid sequence expressed by a cDNA of the invention. In one embodiment, the protein comprises the amino acid sequence of SEQ ID NO:26. The invention additionally provides a composition comprising the protein and a pharmaceutical carrier. The invention also provides a method for using a peptide or protein to screen a plurality of molecules to identify at least one ligand which specifically binds the protein. In one embodiment, the molecules to be screened are selected from agonists, antagonists, antibodies, DNA molecules, transcription factors, RNA molecules, and small drug molecules or compounds. The invention further provides a method of using a peptide or protein to purify a ligand. [0015]
  • The invention provides a method for using the peptide or protein to produce an antibody which specifically binds the protein. A method for preparing polyclonal antibodies comprises immunizing a animal with peptide or protein under conditions to elicit an antibody response, isolating animal antibodies, attaching the peptide or protein to a substrate, contacting the substrate with isolated antibodies under conditions to allow specific binding to the peptide or protein, dissociating the antibodies from the peptide or protein, thereby obtaining purified polyclonal antibodies. A method for preparing monoclonal antibodies comprises immunizing a animal with a peptide or protein under conditions to elicit an antibody response, isolating antibody producing cells from the animal, fusing the antibody producing cells with immortalized cells in culture to form monoclonal antibody producing hybridoma cells, culturing the hybridoma cells, and isolating monoclonal antibodies from culture. [0016]
  • The invention provides purified antibodies which bind specifically to a peptide or protein. The invention also provides a method for using an antibody to detect expression of a peptide or protein in a sample, the method comprising combining the antibody with a sample under conditions for formation of antibody:peptide or protein complexes, and detecting complex formation, wherein complex formation indicates expression of the peptide or protein in the sample. In one aspect, the amount of complex formation when compared to standards is diagnostic of a disorder of the nervous system. [0017]
  • The invention provides a method for immunopurification of a protein comprising attaching an antibody to a substrate, exposing the antibody to a sample containing protein under conditions to allow antibody:protein complexes to form, dissociating the protein from the complex, and collecting purified protein. The invention also provides an array upon which a cDNA encoding a protein, the protein, or an antibody which specifically binds the protein are immobilized. The invention also provides a composition comprising a cDNA, a protein, an antibody, or a ligand which has agonistic or antagonistic activity. [0018]
  • The invention provides an antibody comprising an antigen binding site, wherein the antigen binding site specifically binds to the protein The invention also provides a method for treating a disorder associated with the differential expression of a cDNA that is coexpressed with one or more known atherosclerosis-associated genes in a subject in need, the method comprising the step of administering to the subject in need the antibody in an amount effective for treating the disorder. The invention further provides an immunoconjugate comprising the antigen binding site of the antibody or joined to a therapeutic agent. The invention additionally provides a method for treating a disorder associated with the differential expression of a cDNA that is coexpressed with one or more known atherosclerosis-associated genes in a subject in need, the method comprising the step of administering to the subject in need the immunoconjugate in an amount effective for treating the disorder.[0019]
    • BRIEF DESCRIPTION OF THE SEQUENCE LISTING, FIGURE AND TABLES
  • The Sequence Listing provides exemplary cDNAs associated with atherosclerosis including polynucleotide sequences SEQ ID NOs:1-25 and the polypeptide sequence, SEQ ID NO:26. Each sequence is identified by a sequence identification number (SEQ ID NO). [0020]
  • FIG. 1 shows the cDNA having the nucleic acid sequence of SEQ ID NO:8 which encodes the protein having the amino acid sequence of SEQ ID NO:26. The alignment was produced using MAcDNASIS,PRO software (Hitachi Software Engineering, South San Francisco Calif.).[0021]
  • Table 4 shows the-co-expression values (-log p) between the known atherosclerosis-associated genes (abbreviations as shown in Table 3) and the cDNAs of the invention (SEQ IDs). [0022]
  • Table 5 summarizes the highly significantly coexpression between each cDNA (SEQ ID) and two known atherosclerosis-associated genes ([0023] Gene 1 and Gene 2). P-value and the function or importance of the known atherosclerosis-associated gene are derived from Tables 3 and 4.
  • Table 6 shows transcript images for several of the cDNAs (SEQ ID) of the invention. [0024] Column 1 shows the SEQ ID; column 2, the library name; column 3, the number of cDNAs in the library; column 4, the description of the sample from which the library was constructed; column 5, transcript abundance; and column 6, percent transcript abundance. These sets demonstrate differential expression within experiments using cardiovascular tissues.
  • Table 7 shows microarray data for several of the cDNAs (SEQ ID) of the invention. [0025] Column 1 shows the SEQ ID; column 2, the name of the microarray (GEM) used for the experiment; column 3, the log2 (C5/Cy3 ratio); column 4, the description of the Cy3 sample; and column 5, the description of the Cy5 sample. These data demonstrate differential expression of the cDNAs in experiments using cardiovascular samples.
    • DESCRIPTION OF THE INVENTION
  • It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include the plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a host cell” includes a plurality of such host cells, and a reference to “an antibody” is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth. [0026]
  • Definitions [0027]
  • “Antibody” refers to intact immunoglobulin molecule, a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a recombinant antibody, a humanized antibody, single chain antibodies, a Fab fragment, an F(ab′)[0028] 2 fragment, an Fv fragment; and an antibody-peptide fusion protein.
  • “Antigenic determinant” refers to an antigenic or immunogenic epitope, structural feature, or region of an oligopeptide, peptide, or protein which is capable of inducing formation of an antibody which specifically binds the protein. Biological activity is not a prerequisite for immunogenicity. [0029]
  • “Array” refers to an ordered arrangement of at least two cDNAs, proteins, or antibodies on a substrate. At least one of the cDNAs, proteins, or antibodies represents a control or standard, and the other represents a cDNA, protein, or antibody of diagnostic or therapeutic interest. The arrangement of at least two and up to about 40,000 cDNAs, proteins, or antibodies on the substrate assures that the size and signal intensity of each labeled complex, formed between each cDNA and at least one nucleic acid, each protein and at least one ligand or antibody, or each antibody and at least one protein to which the antibody specifically binds, is individually distinguishable. [0030]
  • “Atherosclerosis-associated cDNA” refers to isolated polynucleotide that exhibits a statistically significant co-expression pattern with known atherosclerosis-associated genes. The cDNAs are represented by SEQ ID NOs:1-25 of the Sequence Listing and the complements of SEQ ID NOs:1-25. They may be of recombinant or synthetic origin, used in its double-stranded or single-stranded form, and combined with vitamins, minerals, carbohydrates, lipids, proteins, other nucleic acids, a pharmaceutical carrier or a labeling moiety to perform a particular activity or form a useful composition. [0031]
  • An “atherosclerotic sample or tissue” may be taken using needles, catheters, or scapels and include vessels including the aorta, arteries, arterioles, endothelial cells, plaque, and blood. The sample may contain nucleic acids, proteins, antibodies, and the like. Additionally the sample may comprise the soluble fraction of a cell preparation; an aliquot of media in which cells were grown; a chromosome, an organelle, or membrane isolated or extracted from a cell; genomic DNA, RNA, or cDNA in solution or bound to a substrate. [0032]
  • A “combination” comprises at least two sequences selected from SEQ ID NOs:1-25 as presented in the Sequence Listing and the complements of SEQ ID NOs:1-25. [0033]
  • “Differential expression” refers to an increased or up-regulated or a decreased or down-regulated expression as detected by absence, presence, or at least two-fold change in the amount of transcribed messenger RNA or translated protein in a sample. [0034]
  • “Disorders associated with atherosclerosis” include angina pectoris, coronary artery disease, myocardial infarction, hypertension, transient cerebral ischemia, mesenteric ischemia, peripheral vascular disease, renal artery stenosis, and stroke. [0035]
  • An “expression profile” is a representation of gene expression in a sample. A nucleic acid expression profile is produced using sequencing, hybridization, or amplification technologies and mRNAs or cDNAs from a sample. A protein expression profile, although time delayed, mirrors the nucleic acid expression profile and uses two-dimensional polyacrylamide electrophoresis (2D-PAGE) and mass spectrophotometry (MS) or western analysis, enzyme-linked immunosorbent assays (ELISAs), fluorescence activated cell sorting (FACS), radioimmunoassays (RIAs), or arrays and labeling moieties or antibodies to detect expression in a sample. The nucleic acids, proteins, or antibodies may be used in solution or attached to a substrate, and their detection is based on methods and labeling moieties well known in the art. [0036]
  • A “hybridization complex” is formed between a cDNA of the invention and a nucleic acid of a sample when the purines of one molecule hydrogen bond with the pyrimidines of the complementary molecule, e.g., 5′-A-G-T-C-3′ base pairs with its complete complement, 3′-T-C-A-G-5′. The degree of complementarity and the use of nucleotide analogs affect the efficiency and stringency of hybridization reactions. [0037]
  • “Identity” as applied to sequences, refers to the quantification (usually percentage) of nucleotide or residue matches between at least two sequences aligned using a standardized algorithm such as Smith-Waterman alignment (Smith and Waterman (1981) J Mol Biol 147:195-197), CLUSTALW (Thompson et al. (1994) Nucleic Acids Res 22:4673-4680), or BLAST2 (Altschul et al. (1997) Nucleic Acids Res 25:3389-340). BLAST2 may be used in a standardized and reproducible way to insert gaps in one of the sequences in order to optimize alignment and to achieve a more meaningful comparison between them. [0038]
  • “Similarity” as applied to proteins uses the same algorithms but takes into account conservative substitutions of nucleotides or residues. [0039]
  • “Isolated or purified” refers to a cDNA or protein that is removed from its natural environment and that is separated from other components with which it is naturally present. [0040]
  • “Genes known to be associated with atherosclerosis” include human 22 kDa smooth muscle protein, calponin (CNN1), pro alpha 1 (I) collagen (COLIA1), collagen alpha-2 type I (COLIA2), collagen alpha-6 type I (COL6A1), procollagen alpha 2(V) (COL5A2), collagen VI alpha-2 (COL6A2), type VI collagen alpha 3 (COL6A3), pro-alpha-i type 3 collagen (COL3A1), pro-alpha-1(V) collagen (COLSA1), matrix Gla protein (MGP), cathepsin K (CSTK), fibrinogen beta chain gene (FBG), pre-pro-von Willebrand factor (VWF), platelet endothelial cell adhesion molecule (PECAM-1), antithrombin III variant (AT3), lipoprotein lipase (LPL), alpha-2-macroglobulin (A2M), apolipoprotein AI (APOA1), apolipoprotein AII (APOA1)[0041] 2, apolipoprotein B-100 (APOB), lipoprotein apoCII (APOC2), pre-apolipoprotein CIII (APOC3), apolipoprotein apo C-IV (APOC4), macrophage scavenger receptor type I (MSR1), human antigen CD36 gene (CD36), serum amyloid P component (SAP), carboxyl ester lipase gene (CEL), paraoxonase 1 (PONI), paraoxonase 2 (PON2), paraoxonase 3 (PON3), perilipin (PLIN), prostaglandin D2 synthase (PTGDS), annexin II/lipocortin II(ANX2), annexin I/lipocortin (ANX1), and secreted protein, acidic and rich in cysteine (SPARC).
  • “Labeling moiety” refers to any reporter molecule whether a visible or radioactive label, stain or dye that can be attached to or incorporated into a cDNA or protein. Visible labels and dyes include but are not limited to anthocyanins, B glucuronidase, BIODIPY, Coomassie blue, Cy3 and Cy5, digoxigenin, FITC, green fluorescent protein, luciferase, spyro red, silver, and the like. Radioactive markers include radioactive forms of hydrogen, iodine, phosphorous, sulfur, and the like. [0042]
  • “Ligand” refers to any agent, molecule, or compound which will bind specifically to a complementary site on a cDNA molecule, a polynucleotide, an epitope or a protein. Such ligands stabilize or modulate the activity of polynucleotides or proteins and may be composed of inorganic or organic substances including nucleic acids, proteins, carbohydrates, fats, and lipids. [0043]
  • “Markers for disorders associated with atherosclerosis” refers to cDNAs, peptides or proteins, and antibodies which are useful in the diagnosis, prognosis, treatment, selection or evaluation of therapies for disorders associated with atherosclerosis. These markers are differentially expressed in samples from subjects predisposed to or manifesting one of these disorders. The known atherosclerosis-associated genes and their contribution and/or function to disorders associated with atherosclerosis are listed in TABLE3. [0044]
  • “Probe” refers to a cDNA of the invention that hybridizes to at least one nucleic acid in a sample. Where targets are single stranded, probes are complementary single strands. Probes can be labeled for use in hybridization reactions including Southern, northern, in situ, dot blot, array, and like technologies or in screening assays. [0045]
  • “Protein” refers to a polypeptide or any portion thereof. An “oligopeptide” is an amino acid sequence from about five residues to about 15 residues that is used as part of a fusion protein to produce an antibody that specifically binds the protein. [0046]
  • “Specific binding” refers to a special and precise interaction between two molecules which is dependent upon their structure, particularly their molecular side groups. For example, the intercalation of a regulatory protein into the major groove of a DNA molecule, the hydrogen bonding along the backbone between two single stranded nucleic acids, or the binding between an epitope of a protein and an agonist, antagonist, or antibody. [0047]
  • “Substrate” refers to any rigid or semi-rigid support to which cDNAs or proteins are bound and includes membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, capillaries or other tubing, plates, polymers, and microparticles with a variety of surface forms including wells, trenches, pins, channels and pores. [0048]
  • A “transcript image” (TI) is a profile of gene transcription activity in a particular tissue at a particular time. TI provides assessment of the relative abundance of expressed transcripts in the cDNA libraries of an EST database as described in U.S. Pat. No. 5,840,484, incorporated herein by reference. [0049]
  • “Variant” refers to molecules that are recognized variations of a polynucleotide or a protein. Splice variants may be determined by BLAST score, wherein the score is at least 100, and most preferably at least 400. Allelic variants have a high percent identity to a polynucleotide and may differ by about three bases per hundred bases. “Single nucleotide polymorphism” (SNP) refers to a change in a single base as a result of a substitution, insertion or deletion. The change may be conservative (purine for purine) or non-conservative (purine to pyrimidine) and may or may not result in a change in an encoded amino acid. [0050]
  • The Method [0051]
  • The present invention encompasses a method for identifying cDNAs that are significantly co-expressed with known atherosclerosis-associated genes. In particular, the method identifies a combination of cDNAs useful in diagnosis, prognosis, treatment, and evaluation of therapies for disorders associated with atherosclerosis. [0052]
  • The method involves identifying cDNAs that are expressed in a plurality of cDNA libraries. These cDNAs include genes of known or unknown function whose expression patterns are compared with genes having a known function and disease association to determine whether a specified coexpression probability threshold is met. Through this comparison, a subset of the cDNAs having a high coexpression probability with the known genes can be identified. [0053]
  • The cDNAs may originate from cDNA libraries derived from a variety of sources including, but not limited to, eukaryotes such as human, mouse, rat, dog, monkey, plant, and yeast; prokaryotes such as bacteria; and viruses. The cDNAs can also be selected from a variety of sequence types including, but not limited to, expressed sequence tags (ESTs), assembled polynucleotide sequences, full length gene coding regions, promoters, introns, enhancers, 5′ untranslated regions, and 3′ untranslated regions. To have statistically significant analytical results, the cDNAs need to be expressed in at least three cDNA libraries. [0054]
  • The cDNA libraries used in the coexpression analysis of the present invention can be obtained from adrenal gland, biliary tract, bladder, blood cells, blood vessels, bone marrow, brain, bronchus, cartilage, chromaffin system, colon, connective tissue, cultured cells, embryonic stem cells, endocrine glands, epithelium, esophagus, fetus, ganglia, heart, hypothalamus, immune system, intestine, islets of Langerhans, kidney, larynx, liver, lung, lymph, muscles, neurons, ovary, pancreas, penis, peripheral nervous system, phagocytes, pituitary, placenta, pleurus, prostate, salivary glands, seminal vesicles, skeleton, spleen, stomach, testis, thymus, tongue, ureter, uterus, and the like. The number of cDNA libraries selected can range from as few as 3 to greater than 10,000. Preferably, the number of the cDNA libraries is greater than 500. [0055]
  • In a preferred embodiment, the cDNAs are assembled from related sequences, such as assembled sequence fragments derived from a single transcript. Assembly of the sequences can be performed using sequences of various types including, but not limited to, ESTs, extensions, or shotgun sequences. In a most preferred embodiment, the cDNAs are derived from human sequences that have been assembled using the algorithm disclosed in U.S. Ser. No. 09/276,534, filed Mar. 25, 1999, incorporated herein by reference. [0056]
  • Experimentally, differential expression of the cDNAs can be evaluated by methods including, but not limited to, differential display by spatial immobilization or by gel electrophoresis, genome mismatch scanning, representational difference analysis, and transcript imaging. Additionally, differential expression can be assessed by microarray technology. These methods may be used alone or in combination. [0057]
  • The known atherosclerosis-associated genes were selected based on their function in pathways associated with atherogenesis, their use as diagnostic or prognostic markers, their behavior in model systems or their use as therapeutic targets. [0058]
  • The procedure for identifying a cDNA that exhibits a statistically significant coexpression pattern with known atherosclerosis-associated genes is as follows. First, the presence or absence of a gene in a cDNA library is defined: a gene is present in a cDNA library when at least one cDNA fragment corresponding to that gene is detected in a cDNA sample taken from the library, and a gene is absent from a library when no corresponding cDNA fragment is detected in the sample. [0059]
  • Second, the significance of gene coexpression is evaluated using a probability method to measure a due-to-chance probability of the coexpression. The probability method can be the Fisher exact test, the chi-squared test, or the kappa test. These tests and examples of their applications are well known in the art and can be found in standard statistics texts (Agresti (1990) [0060] Categorical Data Analysis, John Wiley & Sons, New York N.Y.; Rice (1988) Mathematical Statistics and Data Analysis, Duxbury Press, Pacific Grove Calif.). A Bonferroni correction (Rice, supra, p. 384) can also be applied in combination with one of the probability methods for correcting statistical results of one gene vetsus multiple other genes. In a preferred embodiment, the due-to-chance probability is measured by a Fisher exact test, and the threshold of the due-to-chance probability is set preferably to less than 0.001, more preferably to less than 0.00001.
  • To determine whether two genes, A and B, have similar coexpression patterns, occurrence data vectors can be generated as illustrated in Table 1. The presence of a gene occurring at least once in a library is indicated by a one, and its absence from the library, by a zero. [0061]
    TABLE 1
    Occurrence data for genes A and B
    Library
    1 Library 2 Library 3 . . . Library N
    gene A
    1 1 0 . . . 0
    gene B 1 0 1 . . . 0
  • For a given pair of genes, the occurrence data in Table 1 can be summarized in a 2×2 contingency table. [0062]
    TABLE 2
    Contingency table for co-occurrences of genes A and B
    Gene A present Gene A absent Total
    Gene B present 8 2 10
    Gene B absent 2 18 20
    Total 10 20 30
  • Table 2 presents co-occurrence data for gene A and gene B in a total of 30 libraries. Both gene A and gene B occur 10 times in the libraries. Table 2 summarizes and presents: 1) the number of times gene A and B are both present in a library; 2) the number of times gene A and B are both absent in a library; 3) the number of times gene A is present, and gene B is absent; and 4) the number of times gene B is present, and gene A is absent. The upper left entry is the number of times the two genes co-occur in a library, and the middle right entry is the number of times neither gene occurs in a library. The off diagonal entries are the number of times one gene occurs, and the other does not. Both A and B are present eight times and absent 18 times. Gene A is present, and gene B is absent, two times; and gene B is present, and gene A is absent, two times. The probability (“p-value”) that the above association occurs due to chance as calculated using a Fisher exact test is 0.0003. Associations are generally considered significant if a p-value is less than 0.01 (Agresti, supra; Rice, supra). [0063]
  • This method of estimating the probability for co-expression of two genes makes several assumptions. The method assumes that the libraries are independent and are identically sampled. However, in practical situations, the selected cDNA libraries are not entirely independent, because more than one library may be obtained from a single subject or tissue. Nor are they entirely identically sampled, because different numbers of cDNAs may be sequenced from each library. The number of cDNAs sequenced typically ranges from 5,000 to 10,000 cDNAs per library. In addition, because a Fisher exact co-expression probability is calculated for each gene versus 45,233 other assembled genes, a Bonferroni correction for multiple statistical tests is used. [0064]
  • The Invention [0065]
  • The present invention identifies 25 atherosclerosis-associated cDNAs that exhibit strong association with genes known to be specifically expressed in atherosclerosis. The results presented in Tables 4 and 5 show that the expression of the 25 novel atherosclerosis-associated cDNAs have direct association with the expression of known atherosclerosis-associated genes as described in Table 3 and in the background of the invention. Therefore, the novel atherosclerosis-associated cDNAs can potentially be used in diagnosis, prognosis, treatment or evaluation of therapies fro disorders associated with atherosclerosis. Further, the gene products of the 25 novel atherosclerosis-associated cDNAs are either potential therapeutics or targets for the development of therapeutics against disorders associated with atherosclerosis. [0066]
  • Therefore, in one embodiment, the present invention encompasses a combination comprising a plurality of cDNAs having the nucleic acid sequences of SEQ ID NOs:1-25 or the complements of SEQ ID NOs:1-25. These 25 cDNAs have been shown by the method of the present invention to have statistically significant co-expression with known atherosclerosis-associated genes and with each other. The invention also encompasses a cDNA comprising a polynucleotide having the nucleic acid sequence of SEQ ID NO:8 and the complement thereof. As shown in FIG. 1, SEQ ID NO:8 encodes the protein of SEQ ID NO:26. The invention further encompasses a protein comprising the polypeptide having the amino acid sequence of SEQ ID NO:26. [0067]
  • The protein encoded by SEQ ID NO:8 has 366 amino acids. Motif analyses of SEQ ID NO:26 shows one potential cAMP- and cGMP-dependent protein kinase phosphorylation site at residue S343, two potential casein kinase II phosphorylation sites at residues S179 and T351, and four potential protein kinase C phosphorylation sites at residues T29, S85, T269, and T324. Additionally, SEQ ID NO:26 contains a potential sugar transport protein signature sequence from residues L201 to S217. [0068]
  • cDNAs and Their Uses [0069]
  • cDNAs can be prepared by a variety of synthetic or enzymatic methods well known in the art. cDNAs can be synthesized, in whole or in part, using chemical methods well known in the art (Caruthers et al. (1980) Nucleic Acids Symp Ser (7):215-233). Alternatively, cDNAs can be produced enzymatically or recombinantly, by in vitro or in vivo transcription. [0070]
  • Nucleotide analogs can be incorporated into cDNAs by methods well known in the art. The only requirement is that the incorporated analog must base pair with native purines or pyrimidines. For example, 2,6-diaminopurine can substitute for adenine and form stronger bonds with thymidine than those between adenine and thymidine. A weaker pair is formed when hypoxanthine is substituted for guanine and base pairs with cytosine. Additionally, cDNAs can include nucleotides that have been derivatized chemically or enzymatically. [0071]
  • cDNAs can be synthesized on a substrate. Synthesis on the surface of a substrate may be accomplished using a chemical coupling procedure and a piezoelectric printing apparatus as described by Baldeschweiler et al. (PCT publication WO95/251116). Alternatively, the cDNAs can be synthesized on a substrate surface using a self-addressable electronic device that controls when reagents are added as described by Heller et al. (U.S. Pat. No. 5,605,662). cDNAs can be synthesized directly on a substrate by sequentially dispensing reagents for their synthesis on the substrate surface or by dispensing preformed DNA fragments to the substrate surface. Typical dispensers include a micropipette delivering solution to the substrate with a robotic system to control the position of the micropipette with respect to the substrate. There can be a multiplicity of dispensers so that reagents can be delivered to the reaction regions efficiently. [0072]
  • cDNAs can be immobilized on a substrate by covalent means such as by chemical bonding procedures or UV irradiation. In one method, a cDNA is bound to a glass surface which has been modified to contain epoxide or aldehyde groups. In another method, a cDNA is placed on a polylysine coated surface and UV cross-linked to it as described by Shalon et al. (WO95/35505). In yet another method, a cDNA is actively transported from a solution to a given position on a substrate by electrical means (Heller, supra). cDNAs do not have to be directly bound to the substrate, but rather can be bound to the substrate through a linker group. The linker groups are typically about 6 to 50 atoms long to provide exposure of the attached cDNA. Preferred linker groups include ethylene glycol oligomers, diamines, diacids and the like. Reactive groups on the substrate surface react with a terminal group of the linker to bind the linker to the substrate. The other terminus of the linker is then bound to the cDNA. Alternatively, polynucleotides, plasmids or cells can be arranged on a filter. In the latter case, cells are lysed, proteins and cellular components degraded, and the DNA is coupled to the filter by UV cross-linking. [0073]
  • The cDNAs may be used for a variety of purposes. For example, the combination of the invention may be used on an array. The array, in turn, can be used in high-throughput methods for detecting a related polynucleotide in a sample, screening a plurality of molecules or compounds to identify a ligand, diagnosing a disorder such as diabetes, or inhibiting or inactivating a therapeutically relevant gene related to the cDNA. [0074]
  • When the cDNAs of the invention are employed on an array, the cDNAs are arranged in an ordered fashion so that each cDNA is present at a specified location. Because the cDNAs are at specified locations on the substrate, the hybridization patterns and intensities, which together create a unique, can be interpreted in terms of expression levels of particular genes and can be correlated with a particular metabolic process, condition, disorder, disease, stage of disease, or treatment. [0075]
  • Hybridization [0076]
  • The cDNAs or fragments or complements thereof may be used in various hybridization technologies. The cDNAs may be labeled using a variety of reporter molecules by either PCR, recombinant, or enzymatic techniques. For example, a commercially available vector containing the cDNA is transcribed in the presence of an appropriate polymerase, such as T7 or SP6 polymerase, and at least one labeled nucleotide. Commercial kits are available for labeling and cleanup of such cDNAs. Radioactive (Amersham Biosciences (APB), Piscataway N.J.), fluorescent (Qiagen-Operon, Alameda Calif.), and chemiluminescent labeling (Promega, Madison Wis.) are well known in the art. [0077]
  • A cDNA may represent the complete coding region of an mRNA or be designed or derived from unique regions of the mRNA or genomic molecule, an intron, a 3′ untranslated region, or from a conserved motif. The cDNA is at least 18 contiguous nucleotides in length and is usually single stranded. Such a cDNA may be used under hybridization conditions that allow binding only to an identical sequence, a naturally occurring molecule encoding the same protein, or an allelic variant. Discovery of related human and mammalian sequences may also be accomplished using a pool of degenerate cDNAs and appropriate hybridization conditions. Generally, a cDNA for use in Southern or northern hybridizations may be from about 400 to about 6000 nucleotides long. Such cDNAs have high binding specificity in solution-based or substrate-based hybridizations. An oligonucleotide, a fragment of the cDNA, may be used to detect a polynucleotide in a sample using PCR. [0078]
  • The stringency of hybridization is determined by G+C content of the cDNA, salt concentration, and temperature. In particular, stringency is increased by reducing the concentration of salt or raising the hybridization temperature. In solutions used for some membrane based hybridizations, addition of an organic solvent such as formamide allows the reaction to occur at a lower temperature. Hybridization may be performed with buffers, such as 5×saline sodium citrate (SSC) with 1% sodium dodecyl sulfate (SDS) at 60° C., that permit the formation of a hybridization complex between nucleic acid sequences that contain some mismatches. Subsequent washes are performed with buffers such as 0.2×SSC with 0.1% SDS at either 45° C. (medium stringency) or 65°-68° C. (high stringency). At high stringency, hybridization complexes will remain stable only where the nucleic acids are completely complementary. In some membrane-based hybridizations, preferably 35% or most preferably 50%, formamide may be added to the hybridization solution to reduce the temperature at which hybridization is performed. Background signals may be reduced by the use of detergents such as Sarkosyl or TRITON X-100 (Sigma-Aldrich, St. Louis Mo.) and a blocking agent such as denatured salmon sperm DNA. Selection of components and conditions for hybridization are well known to those skilled in the art and are reviewed in Ausubel et al. (1997[0079] , Short Protocols in Molecular Biology, John Wiley & Sons, New York N.Y., Units 2.8-2.11, 3.18-3.19 and 4.6-4.9).
  • Dot-blot, slot-blot, low density and high density arrays are prepared and analyzed using methods known in the art. cDNAs from about 18 consecutive nucleotides to about 5000 consecutive nucleotides in length are contemplated by the invention and used in array technologies. The preferred number of cDNAs on an array is at least about 100,000, a more preferred number is at least about 40,000, an even more preferred number is at least about 10,000, and a most preferred number is at least about 600 to about 800. The array may be used to monitor the expression level of large numbers of genes simultaneously and to identify genetic variants, mutations, and SNPs. Such information may be used to determine gene function; to understand the genetic basis of a disorder; to diagnose a disorder; and to develop and monitor the activities of therapeutic agents being used to control or cure a disorder. (See, e.g., U.S. Pat. No. 5,474,796; WO95/11995; WO95/35505; U.S. Pat. No. 5,605,662; and U.S. Pat. No. 5,958,342.) [0080]
  • Screening and Purification Assays Using cDNAs [0081]
  • A cDNA may be used to screen a library or a plurality of molecules or compounds for a ligand which specifically binds the cDNA. Ligands may be DNA molecules, RNA molecules, peptide nucleic acid molecules, peptides, proteins such as transcription factors, promoters, enhancers, repressors, and other proteins that regulate replication, transcription, or translation of the polynucleotide in the biological system. The assay involves combining the cDNA or a fragment thereof with the molecules or compounds under conditions that allow specific binding and detecting the bound cDNA to identify at least one ligand that specifically binds the cDNA. [0082]
  • In one embodiment, the cDNA may be incubated with a library of isolated and purified molecules or compounds and binding activity determined by methods such as a gel-retardation assay (U.S. Pat. No. 6,010,849) or a reticulocyte lysate transcriptional assay. In another embodiment, the cDNA may be incubated with nuclear extracts from biopsied and/or cultured cells and tissues. Specific binding between the cDNA and a molecule or compound in the nuclear extract is initially determined by gel shift assay. Protein binding may be confirmed by raising antibodies against the protein and adding the antibodies to the gel-retardation assay where specific binding will cause a supershift in the assay. [0083]
  • In another embodiment, the cDNA may be used to purify a molecule or compound using affinity chromatography methods well known in the art. In one embodiment, the cDNA is chemically reacted with cyanogen bromide groups on a polymeric resin or gel. Then a sample is passed over and reacts with or binds to the cDNA. The molecule or compound which is bound to the cDNA may be released from the cDNA by increasing the salt concentration of the flow-through medium and collected. [0084]
  • The cDNA may be used to purify a ligand from a sample. A method for using a cDNA to purify a ligand would involve combining the cDNA or a fragment thereof with a sample under conditions to allow specific binding, recovering the bound cDNA, and using an appropriate agent to separate the cDNA from the purified ligand. [0085]
  • Protein Production and Uses [0086]
  • The full length cDNAs or fragments thereof may be used to produce purified proteins using recombinant DNA technologies described herein and taught in Ausubel (supra; Units 16.1-16.62). One of the advantages of producing proteins by these procedures is the ability to obtain highly-enriched sources of the proteins thereby simplifying purification procedures. [0087]
  • The proteins may contain amino acid substitutions, deletions or insertions made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. Such substitutions may be conservative in nature when the substituted residue has structural or chemical properties similar to the original residue (e.g., replacement of leucine with isoleucine or valine) or they may be nonconservative when the replacement residue is radically different (e.g., a glycine replaced by a tryptophan). Computer programs included in LASERGENE software (DNASTAR, Madison Wis.) and algorithms included in RasMol software (University of Massachusetts, Amherst Mass.) may be used to help determine which and how many amino acid residues in a particular portion of the protein may be substituted, inserted, or deleted without abolishing biological or immunological activity. [0088]
  • Expression of Encoded Proteins [0089]
  • Expression of a particular cDNA may be accomplished by cloning the cDNA into a vector and transforming this vector into a host cell. The cloning vector used for the construction of cDNA libraries in the LIFESEQ databases (Incyte Genomics, Palo Alto Calif.) may also be used for expression. Such vectors usually contain a promoter and a polylinker useful for cloning, priming, and transcription. An exemplary vector may also contain the promoter for β-galactosidase, an amino-terminal methionine and the subsequent seven amino acid residues of β-galactosidase. The vector may be transformed into competent [0090] E. coli cells. Induction of the isolated bacterial strain with isopropylthiogalactoside (IPTG) using standard methods will produce a fusion protein that contains an N terminal methionine, the first seven residues of β-galactosidase, about 15 residues of linker, and the protein encoded by the cDNA.
  • The cDNA may be shuttled into other vectors known to be useful for expression of protein in specific hosts. Oligonucleotides containing cloning sites and fragments of DNA sufficient to hybridize to stretches at both ends of the cDNA may be chemically synthesized by standard methods. These primers may then be used to amplify the desired fragments by PCR. The fragments may be digested with appropriate restriction enzymes under standard conditions and isolated using gel electrophoresis. Alternatively, similar fragments are produced by digestion of the cDNA with appropriate restriction enzymes and filled in with chemically synthesized oligonucleotides. Fragments of the coding sequence from more than one gene may be ligated together and expressed. [0091]
  • Signal sequences that dictate secretion of soluble proteins are particularly desirable as component parts of a recombinant sequence. For example, a chimeric protein may be expressed that includes one or more additional purification-facilitating domains. Such domains include, but are not limited to, metal-chelating domains that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex, Seattle Wash.). The inclusion of a cleavable-linker sequence such as ENTEROKINASEMAX (Invitrogen, San Diego Calif.) between the protein and the purification domain may also be used to recover the protein. [0092]
  • Suitable host cells may include, but are not limited to, mammalian cells such as Chinese Hamster Ovary (CHO) and human 293 cells, insect cells such as Sf9 cells, plant cells such as [0093] Nicotiana tabacum, yeast cells such as Saccharomyces cerevisiae, and bacteria such as E. coli. For each of these cell systems, a useful vector may also include an origin of replication and one or two selectable markers to allow selection in bacteria as well as in a transformed eukaryotic host. Vectors for use in eukaryotic host cells may require the addition of 3′ poly(A) tail if the cDNA lacks poly(A).
  • Additionally, the vector may contain promoters or enhancers that increase gene expression. Many promoters are known and used in the art. Most promoters are host specific and exemplary promoters includes SV40 promoters for CHO cells; T7 promoters for bacterial hosts; viral promoters and enhancers for plant cells; and PGH promoters for yeast. Adenoviral vectors with the rous sarcoma virus enhancer or retroviral vectors with long terminal repeat promoters may be used to drive protein expression in mammalian cell lines. Once homogeneous cultures of recombinant cells are obtained, large quantities of secreted soluble protein may be recovered from the conditioned medium and analyzed using chromatographic methods well known in the art. An alternative method for the production of large amounts of secreted protein involves the transformation of mammalian embryos and the recovery of the recombinant protein from milk produced by transgenic cows, goats, sheep, and the like. [0094]
  • In addition to recombinant production, proteins or portions thereof may be produced manually, using solid-phase techniques (Stewart et al. (1969) [0095] Solid-Phase Peptide Synthesis, W H Freeman, San Francisco Calif.; Merrifield (1963) J Am Chem Soc 5:2149-2154), or using machines such as the 431A peptide synthesizer (Applied Biosystems (ABI), Foster City Calif.). Proteins produced by any of the above methods may be used as pharmaceutical compositions to treat disorders associated with null or inadequate expression of the genomic sequence.
  • Screening and Purification Assays Using Proteins [0096]
  • A protein or a portion thereof encoded by the cDNA may be used to screen a library or a plurality of molecules or compounds for a ligand with specific binding affinity or to purify a molecule or compound from a sample. The protein or portion thereof employed in such screening may be free in solution, affixed to an abiotic or biotic substrate, or located intracellularly. For example, viable or fixed prokaryotic host cells that are stably transformed with recombinant nucleic acids that have expressed and positioned a protein on their cell surface can be used in screening assays. The cells are screened against a library or a plurality of ligands and the specificity of binding or formation of complexes between the expressed protein and the ligand may be measured. The ligands may be agonists, antagonists, antibodies, DNA molecules, enhancers, small drug molecules, immunoglobulins, inhibitors, mimetics, peptide nucleic acid molecules, peptides, pharmaceutical agents, proteins, and regulatory proteins, repressors, RNA molecules, ribozymes, transcription factors, or any other test molecule or compound that specifically binds the protein. An exemplary assay involves combining the mammalian protein or a portion thereof with the molecules or compounds under conditions that allow specific binding and detecting the bound protein to identify at least one ligand that specifically binds the protein. [0097]
  • This invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding the protein specifically compete with a test compound capable of binding to the protein or oligopeptide or fragment thereof. One method for high throughput screening using very small assay volumes and very small amounts of test compound is described in U.S. Pat. No. 5,876,946. Molecules or compounds identified by screening may be used in a model system to evaluate their toxicity, diagnostic, or therapeutic potential. [0098]
  • The protein may be used to purify a ligand from a sample. A method for using a protein to purify a ligand would involve combining the protein or a portion thereof with a sample under conditions to allow specific binding, recovering the bound protein, and using an appropriate chaotropic agent to separate the protein from the purified ligand. [0099]
  • Production of Antibodies [0100]
  • A protein encoded by a cDNA of the invention may be used to produce specific antibodies. Antibodies may be produced using an oligopeptide or a portion of the protein with inherent immunological activity. Methods for producing antibodies include: 1) injecting an animal, usually goats, rabbits, or mice, with the protein, or an antigenically-effective portion or an oligopeptide thereof, to induce an immune response; 2) engineering hybridomas to produce monoclonal antibodies; 3) inducing in vivo production in the lymphocyte population; or 4) screening libraries of recombinant immunoglobulins. Recombinant immunoglobulins may be produced as taught in U.S. Pat. No. 4,816,567. [0101]
  • Antibodies produced using the proteins of the invention are useful for the diagnosis of prepathologic disorders as well as the diagnosis of chronic or acute diseases characterized by abnormalities in the expression, amount, or distribution of the protein. A variety of protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies specific for proteins are well known in the art. Immunoassays typically involve the formation of complexes between a protein and its specific binding molecule or compound and the measurement of complex formation. Immunoassays may employ a two-site, monoclonal-based assay that utilizes monoclonal antibodies reactive to two noninterfering epitopes on a specific protein or a competitive binding assay (Pound (1998) [0102] Immunochemical Protocols, Humana Press, Totowa N.J.).
  • Immunoassay procedures may be used to quantify expression of the protein in cell cultures, in subjects with a particular disorder or in model animal systems under various conditions. Increased or decreased production of proteins as monitored by immunoassay may contribute to knowledge of the cellular activities associated with developmental pathways, engineered conditions or diseases, or treatment efficacy. The quantity of a given protein in a given tissue may be determined by performing immunoassays on freeze-thawed detergent extracts of biological samples and comparing the slope of the binding curves to binding curves generated by purified protein. [0103]
  • Antibody Arrays [0104]
  • In an alternative to yeast two hybrid system analysis of proteins, an antibody array can be used to study protein-protein interactions and phosphorylation. A variety of protein ligands are immobilized on a membrane using methods well known in the art. The array is incubated in the presence of cell lysate until protein:antibody complexes are formed. Proteins of interest are identified by exposing the membrane to an antibody specific to the protein of interest. In the alternative, a protein of interest is labeled with digoxigenin (DIG) and exposed to the membrane; then the membrane is exposed to anti-DIG antibody which reveals where the protein of interest forms a complex. The identity of the proteins with which the protein of interest interacts is determined by the position of the protein of interest on the membrane. [0105]
  • Antibody arrays can also be used for high-throughput screening of recombinant antibodies. Bacteria containing antibody genes are robotically-picked and gridded at high density (up to 18,342 different double-spotted clones) on a filter. Up to 15 antigens at a time are used to screen for clones to identify those that express binding antibody fragments. These antibody arrays can also be used to identify proteins which are differentially expressed in samples (de Wildt et al. (2000) Nature Biotechnol 18:989-94). [0106]
  • Labeling of Molecules for Assay [0107]
  • A wide variety of reporter molecules and conjugation techniques are known by those skilled in the art and may be used in various cDNA, polynucleotide, protein, peptide or antibody assays. Synthesis of labeled molecules may be achieved using commercial kits for incorporation of a labeled nucleotide such as [0108] 32P-dCTP, Cy3-dCTP or Cy5-dCTP or amino acid such as 35S-methionine. Polynucleotides, cDNAs, proteins, or antibodies may be directly labeled with a reporter molecule by chemical conjugation to amines, thiols and other groups present in the molecules using reagents such as BIODIPY or FITC (Molecular Probes, Eugene Oreg.).
  • The proteins and antibodies may be labeled for purposes of assay by joining them, either covalently or noncovalently, with a reporter molecule that provides for a detectable signal. A wide variety of labels and conjugation techniques are known and have been reported in the scientific and patent literature including, but not limited to U.S. Pat. No. 3,817,837; U.S. Pat. No. 3,850,752; U.S. Pat. No. 3,939,350; U.S. Pat. No. 3,996,345; U.S. Pat. No. 4,277,437; U.S. Pat. No. 4,275,149; and U.S. Pat. No. 4,366,241. [0109]
  • Diagnostics [0110]
  • The cDNAs, or fragments thereof, may be used to detect and quantify differential gene expression; absence, presence, or excess expression of mRNAs; or to monitor mRNA levels during therapeutic intervention. Disorders associated with atherosclerosis include angina pectoris, coronary artery disease, myocardial infarction, hypertension, transient cerebral ischemia, mesenteric ischemia, peripheral vascular disease, renal artery stenosis, and stroke. These cDNAs can also be utilized as markers of treatment efficacy against the disorders noted above and other disorders, conditions, and diseases over a period ranging from several days to months. The diagnostic assay may use hybridization or amplification technology to compare gene expression in a biological sample from a patient to standard samples in order to detect altered gene expression. Qualitative or quantitative methods for this comparison are well known in the art. [0111]
  • For example, the cDNA may be labeled by standard methods and added to a biological sample from a patient under conditions for hybridization complex formation. After an incubation period, the sample is washed and the amount of label (or signal) associated with hybridization complexes is quantified and compared with a standard value. If the amount of label in the patient sample is significantly altered in comparison to the standard value, then the presence of the associated condition, disease or disorder is indicated. [0112]
  • In order to provide a basis for the diagnosis of a condition, disease or disorder associated with gene expression, a normal or standard expression profile is established. This may be accomplished by combining a biological sample taken from normal subjects, either animal or human, with a probe under conditions for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained using normal subjects with values from an experiment in which a known amount of a purified target sequence is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a particular condition, disease, or disorder. Deviation from standard values toward those associated with a particular condition is used to diagnose that condition. [0113]
  • Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies and in clinical trial or to monitor the treatment of an individual patient. Once the presence of a condition is established and a treatment protocol is initiated, diagnostic assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in a normal subject. The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months. [0114]
  • Gene Expression Profiles [0115]
  • A gene expression profile comprises a plurality of cDNAs and a plurality of detectable hybridization complexes, wherein each complex is formed by hybridization of one or more probes to one or more complementary nucleic acids in a sample. The cDNAs of the invention are used as elements on a array to analyze gene expression profiles. In one embodiment, the array is used to monitor the progression of disease. Researchers or clinicians can catalog the differences in gene expression between healthy and diseased tissues or cells. By analyzing changes in patterns of gene expression, disease can be diagnosed at earlier stages before the patient is symptomatic. The invention can be used to formulate a prognosis and to design a treatment regimen. The invention can also be used to monitor the efficacy of treatment. For treatments with known side effects, the array is employed to improve the treatment regimen. A dosage is established that causes a change in genetic expression patterns indicative of successful treatment. Expression patterns associated with the onset of undesirable side effects are avoided. This approach may be more sensitive and rapid than waiting for the patient to show inadequate improvement, or to manifest side effects, before altering the course of treatment. [0116]
  • Experimentally, expression profiles can also be evaluated by methods including, but not limited to, differential display by spatial immobilization or by gel electrophoresis, labeling with radionuclide and quantification using a scintillation counter, genome mismatch scanning, representational difference analysis, transcript imaging, quantitative PCR, and by protein or antibody arrays. Expression profiles produced by these methods may be contrasted with expression profiles produced using normal or diseased tissues. Of note is the correspondence between mRNA and protein expression has been discussed by Zweiger (2001[0117] , Transducing the Genome. McGraw-Hill, San Francisco, Calif.) and Glavas et al. (2001; T cell activation upregulates cyclic nucleotide phosphodiesterases 8A1 and 7A3, Proc Natl Acad Sci 98:6319-6342) among others.
  • In another embodiment, animal models which mimic a human disease can be used to produce expression profiles associated with a particular condition, disorder or disease; or treatment of the condition, disorder or disease. Novel treatment regimens may be tested in these animal models using arrays to establish and then follow expression profiles over time. In addition, arrays may be used with cell cultures or tissues removed from animal models to rapidly screen large numbers of candidate drug molecules, looking for ones that produce an expression profile similar to those of known therapeutic drugs, with the expectation that molecules with the same expression profile will likely have similar therapeutic effects. Thus, the invention provides the means to rapidly determine the molecular mode of action of a drug. [0118]
  • Assays Using Antibodies [0119]
  • Antibodies directed against antigenic determinants of a protein encoded by a cDNA of the invention may be used in assays to quantify the amount of protein found in a particular human cell. Such assays include methods utilizing the antibody and a label to detect expression level under normal or disease conditions. The antibodies may be used with or without modification, and labeled by joining them, either covalently or noncovalently, with a labeling moiety. [0120]
  • Protocols for detecting and measuring protein expression using either polyclonal or monoclonal antibodies are well known in the art. Examples include, but are not limited to, western analysis, ELISA, RIA, FACS, and arrays. Such immunoassays typically involve the formation of complexes between the protein and its specific antibody and the measurement of such complexes. These assays are specifically described in Pound (supra). [0121]
  • Therapeutics [0122]
  • The cDNAs and fragments thereof can be used in gene therapy. cDNAs can be delivered ex vivo to target cells, such as cells of bone marrow. Once stable integration and transcription and or translation are confirmed, the bone marrow may be reintroduced into the subject. Expression of the protein encoded by the cDNA may correct a disorder associated with mutation of a normal sequence, reduction or loss of an endogenous target protein, or overepression of an endogenous or mutant protein. Alternatively, cDNAs may be delivered in vivo using vectors such as retrovirus, adenovirus, adeno-associated virus, herpes simplex virus, and bacterial plasmids. Non-viral methods of gene delivery include cationic liposomes, polylysine conjugates, artificial viral envelopes, and direct injection of DNA (Anderson (1998) Nature 392:25-30; Dachs et al. (1997) Oncol Res 9:313-325; Chu et al. (1998) J Mol Med 76(3-4):184-192; Weiss et al. (1999) Cell Mol Life Sci 55(3):334-358; Agrawal (1996) [0123] Antisense Therapeutics, Humana Press, Totowa N.J.; and August et al. (1997) Gene Therapy (Advances in Pharmacology, Vol. 40), Academic Press, San Diego Calif.).
  • In addition, expression of a particular protein can be regulated through the specific binding of a fragment of a cDNA to a genomic sequence or an mRNA which encodes the protein or directs its transcription or translation. The cDNA can be modified or derivatized to any RNA-like or DNA-like material including peptide nucleic acids, branched nucleic acids, and the like. These sequences can be produced biologically by transforming an appropriate host cell with a vector containing the sequence of interest. [0124]
  • Molecules which regulate the activity of the cDNA or encoded protein are useful as therapeutics for diabetes mellitus, obesity, hypertension, atherosclerosis, polycystic ovarian syndrome, and cancers including breast, prostate, and colon. Such molecules include agonists which increase the expression or activity of the polynucleotide or encoded protein, respectively; or antagonists which decrease expression or activity of the polynucleotide or encoded protein, respectively. In one aspect, an antibody which specifically binds the protein may be used directly as an antagonist or indirectly as a delivery mechanism for bringing a pharmaceutical agent to cells or tissues which express the protein. [0125]
  • Additionally, any of the proteins, or their ligands, or complementary nucleic acid sequences may be administered as pharmaceutical compositions or in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to affect the treatment or prevention of the conditions and disorders associated with an immune response. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects. Further, the therapeutic agents may be combined with pharmaceutically-acceptable carriers including excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration used by doctors and pharmacists may be found in the latest edition of Remington's Pharmaceutical Sciences (Mack Publishing, Easton Pa.). [0126]
  • Model Systems [0127]
  • Animal models may be used as bioassays where they exhibit a phenotypic response similar to that of humans and where exposure conditions are relevant to human exposures. Mammals are the most common models, and most infectious agent, cancer, drug, and toxicity studies are performed on rodents such as rats or mice because of low cost, availability, lifespan, reproductive potential, and abundant reference literature. Inbred and outbred rodent strains provide a convenient model for investigation of the physiological consequences of underexpression or overexpression of genes of interest and for the development of methods for diagnosis and treatment of diseases. A mammal inbred to overexpress a particular gene (for example, secreted in milk) may also serve as a convenient source of the protein expressed by that gene. [0128]
  • Transgenic Animal Models [0129]
  • Transgenic rodents that overexpress or underexpress a gene of interest may be inbred and used to model human diseases or to test therapeutic or toxic agents. (See, e.g., U.S. Pat. No. 5,175,383 and U.S. Pat. No. 5,767,337.) In some cases, the introduced gene may be activated at a specific time in a specific tissue type during fetal or postnatal development. Expression of the transgene is monitored by analysis of phenotype, of tissue-specific mRNA expression, or of serum and tissue protein levels in transgenic animals before, during, and after challenge with experimental drug therapies. [0130]
  • Embryonic Stem Cells [0131]
  • Embryonic (ES) stem cells isolated from rodent embryos retain the potential to form embryonic tissues. When ES cells such as the mouse 129/SvJ cell line are placed in a blastocyst from the C57BL/6 mouse strain, they resume normal development and contribute to tissues of the live-born animal. ES cells are preferred for use in the creation of experimental knockout and knockin animals. The method for this process is well known in the art and the steps are: the cDNA is introduced into a vector, the vector is transformed into ES cells, transformed cells are identified and microinjected into mouse cell blastocysts, blastocysts are surgically transferred to pseudopregnant dams. The resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains. [0132]
  • Knockout Analysis [0133]
  • In gene knockout analysis, a region of a gene is enzymatically modified to include a non-natural intervening sequence such as the neomycin phosphotransferase gene (neo; Capecchi (1989) Science 244:1288-1292). The modified gene is transformed into cultured ES cells and integrates into the endogenous genome by homologous recombination. The inserted sequence disrupts transcription and translation of the endogenous gene. [0134]
  • Knockin Analysis [0135]
  • ES cells can be used to create knockin humanized animals or transgenic animal models of human diseases. With knockin technology, a region of a human gene is injected into animal ES cells, and the human sequence integrates into the animal cell genome. Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on the progression and treatment of the analogous human condition. [0136]
  • As described herein, the uses of the cDNAs, provided in the Sequence Listing of this application, and their encoded proteins are exemplary of known techniques and are not intended to reflect any limitation on their use in any technique that would be known to the person of average skill in the art. Furthermore, the cDNAs provided in this application may be used in molecular biology techniques that have not yet been developed, provided the new techniques rely on properties of nucleotide sequences that are currently known to the person of ordinary skill in the art, e.g., the triplet genetic code, specific base pair interactions, and the like. Likewise, reference to a method may include combining more than one method for obtaining or assembling full length cDNA sequences that will be known to those skilled in the art. It is also to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary. It is also understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. The examples below are provided to illustrate the subject invention and are not included for the purpose of limiting the invention. [0137]
    • EXAMPLES
  • It is to be understood that this invention is not limited to the particular devices, machines, materials and methods described. Although particular embodiments are described, equivalent embodiments may be used to practice the invention. The described embodiments are provided to illustrate the invention and are not intended to limit the scope of the invention which is limited only by the appended claims. [0138]
  • I cDNA Library Construction [0139]
  • The cDNA library SMCCNOS01 was selected as an example to demonstrate the construction of cDNA libraries from which the cDNAs co-expressed with known atherosclerosis-associated genes were derived. The SMCCNOS01 subtracted coronary artery smooth muscle cell library was constructed using 7.56×10[0140] 6 clones from the SMCCNOT02 library and was subjected to two rounds of subtraction hybridization for 48 hours with 6.12×106 clones from SMCCNOT01.
  • The SMCCNOT02 library was constructed using RNA isolated from coronary artery smooth muscle cells removed from a 3-year-old Caucasian male. The cells were treated for 20 hours with TNFα and IL-1β at 10 ng/ml each. The SMCCNOT01 was constructed using RNA isolated from untreated coronary artery smooth muscle cells from the same donor. Subtractive hybridization conditions were based on the methodologies of Swaroop et al. (1991; Nucleic Acids Res 19:1954) and Bonaldo et al. (1996; Genome Research 6:791). [0141]
  • For both cDNA libraries, SMCCNOT01 and SMCCNOT02, the frozen coronary artery smooth muscle cells (50-100 mg) were homogenized in GTC buffer (4.0M guanidine thiocyanate, 0.1M Tris-HCl pH 7.5, 1% 2-mercaptoethanol). Two volumes of binding buffer (0.4M LiCl, 0.1M Tris-HCl pH 7.5, 0.02M EDTA) were added, and the resulting mixture was vortexed at 13,000 rpm. The supernatant was removed and combined with oligo d(T)[0142] 25 bound streptavidin particles (MPG). After rotation at room temperature, the mRNA-oligo d(T)25 bound streptavidin particles were separated from the supernatant, washed twice with hybridization buffer 1 (0.15M NaCl, 0.01M Tris-HCl pH 8.0, 1 mM EDTA, 0.1% lauryl sarcosinate) using magnetic separation at each step to remove the supernatant from the particles. Bound mRNA was eluted from the particles with release solution and heated to 65° C. The supernatant containing eluted mRNA was magnetically separated from the particles and used to construct the cDNA libraries.
  • The RNA was used according to the recommended protocols in the SUPERSCRIPT plasmid system (Invitrogen). The cDNAs were fractionated on a SEPHAROSE CLIB column (APB), and those cDNAs exceeding 400 bp were ligated into pINCY plasmid (Incyte Genomics, Palo Alto Calif.). Recombinant plasmids were transformed into DH5α competent cells or ELECTROMAX cells (Invitrogen). [0143]
  • II Isolation and Sequencing of cDNA Clones [0144]
  • Plasmid DNA was released from the cells and purified using the REAL Prep 96 plasmid kit (Qiagen, Valencia Calif.). The recommended protocol was employed except for the following changes: 1) the bacteria were inoculated into 1 ml of sterile TERRIFIC BROTH (BD Biosciences, San Jose Calif.) with carbenicillin at 25 mg/l and glycerol at 0.4%; 2) the cells were cultured for 19 hours and then lysed with 0.3 ml of lysis buffer; 3) following isopropanol precipitation, the plasmid DNA pellet was resuspended in 0.1 ml distilled water, and 4) the samples were transferred to a 96-well block for storage at 4° C. [0145]
  • The cDNAs were prepared using a MICROLAB 2200 system (Hamilton, Reno Nev.) in combination with the DNA ENGINE thermal cycler (MJ Research, Watertown Mass.). cDNAs were sequenced by the method of Sanger et al. (1975, J Mol Biol 94:441-446) using PRISM 377 (ABI) or MEGABACE 1000 sequencing systems (APB). [0146]
  • Most of the sequences were sequenced using standard protocols and kits (ABI) at solution volumes of 0.25×-1.0×concentrations. In the alternative, some of the sequences disclosed herein were sequenced using solutions and dyes from APB. [0147]
  • III Selection, Assembly, and Characterization of Sequences [0148]
  • The sequences used for co-expression analysis were assembled from EST sequences, 5′ and 3′ longread sequences, and full length coding sequences. The cDNAs claimed herein were expressed in at least three libraries. [0149]
  • The assembly process is described as follows. EST sequence chromatograms were processed and verified. Quality scores were obtained using PHRED (Ewing et al. (1998) Genome Res 8:175-185; Ewing and Green (1998) Genome Res 8:186-194), and edited sequences were loaded into a relational database management system (RDBMS). The sequences were clustered using BLAST with a product score of 50. All clusters of two or more sequences created a bin which represents one transcribed gene. [0150]
  • Assembly of the component sequences within each bin was performed using a modification of Phrap, a publicly available program for assembling DNA fragments (Green, P. University of Washington, Seattle Wash.). Bins that showed 82% identity from a local pair-wise alignment between any of the consensus sequences were merged. [0151]
  • Bins were annotated by screening the consensus sequence in each bin against public databases, such as GBpri and GenPept from NCBI. The annotation process involved a FASTn screen against the GBpri database in GenBank. Those hits with a percent identity of greater than or equal to 75% and an alignment length of greater than or equal to 100 base pairs were recorded as homolog hits. The residual unannotated sequences were screened by FASTx against GenPept. Those alignments with an E value of less than or equal to 10[0152] −8 were recorded as homologs.
  • Sequences were then reclustered using BLASTn and Cross-Match, a program for rapid amino acid and nucleic acid sequence comparison and database search (Green, supra), sequentially. Any BLAST alignment between a sequence and a consensus sequence with a score greater than 150 was realigned using cross-match. The sequence was added to the bin whose consensus sequence gave the highest Smith-Waterman score (Smith et al. (1992) Protein Engineering 5:35-51) amongst local alignments with at least 82% identity. Non-matching sequences were moved into new bins, and assembly processes were repeated. [0153]
  • IV Coexpression Analyses of Atherosclerosis-Associated Genes [0154]
  • Known atherosclerosis-associated genes were selected to identify the cDNAs that are closely associated with atherosclerosis. The known atherosclerosis-associated genes which were used in this analysis all occur within the LIFESEQ Gold database (Incyte Genomics), and brief descriptions of their functions as they have been reported in the literature are listed in Table 3. [0155]
    TABLE 3
    Descriptions of Known Atherosclerosis-Associated Genes
    GENE DESCRIPTION AND REFERENCES
    Human 22kDa Smooth muscle cell-specific gene which is down-regulated during smooth
    smooth muscle muscle cell dedifferentiation as part of atherogenic process (Sobue et al. (1998)
    protein (SM22) Horm Res 50(S2):15-24; Sobue et al. (1999) Mol Cell Biochem 190:105-18)
    calponin (CNN1) Calponin is smooth muscle-specific and may mediate smooth muscle
    contractility through binding of the amino-terminal end of the myosin regulatory
    light chain. Involved in phenotypic modulation of smooth muscle cells, a feature
    of atherosclerosis (Szymanski et al.(1999) Biochemistry 38:3778-84)
    pro alpha 1(I) Member of family of fibrous structural proteins. Most abundant structural
    collagen (COL1A1) component of the extracellular matrix. Secreted as procollagen and converted
    to collagen by matrix metalloproteinases. Collagens are important in
    atherosclerosis for promoting platelet aggregation and for providing sites for
    platelet adhesion to the vessel wall (Wen et al. (1999) Arterioscler Thromb
    Vasc Biol 19:519-24)
    collagen alpha-2 type see COL1A1 above
    I (COL1A2)
    COL6A1 see COL1A1 above
    procollagen alpha see COL1A1 above
    2(V) (COL5A2)
    collagen VI alpha-2 see COL1A1 above
    (COL6A2)
    type VI collagen see COL1A1 above
    alpha3 (COL6A3)
    pro-alpha-1 type 3 see COL1A1 above
    collagen (COL3A1)
    pro-alpha-1 (V) see COL1A1 above
    collagen (COL3A1)
    matrix Gla protein Role in active calcification of vascular smooth muscle cells, suggested by
    (MGP) expression study on VSMC in vitro differentiation study. Calcifying phenotype
    associated with high MGP levels. MGP knockout mice develop to term, but die
    up to 2 months after birth due to extensive calcification of the arteries, causing
    blood vessel rupture (Luo et al. (1997) Nature 386:78-81; Mori et al. (1998)
    FEES Lett 433:19-22)
    cathepsin K (CTSK) Nonmetalloenzyme, potent elastase present in advanced atherosclerotic
    plaques. Contributes to the breakdown of components of vascular extracellular
    matrix, reducing tensile strength, increasing plaque vulnerability (Sukhova et
    al.(1998) J Clin Invest 102:576-83)
    fibrinogen beta chain Component of fibrin in the extracellular matrix. Fibrin deposition is an integral
    gene (FGB) part of advanced atherosclerotic lesion development. Variation at the beta
    fibrinogen locus associated with peripheral atherosclerosis (Sueishi et al. (1998)
    Semin Thromb Hemost 24:255-260; Fowkes et al.(1992) Lancet 339:693-696)
    pre-pro-von Blood glycoprotein involved in normal hemostasis. Mediates adhesion of
    Willebrand factor platelets to sites of vascular damage. Also acts as a cofactor in factor VIII
    (VWF) activity in blood coagulation. Increased levels of VWF are found in
    atherosclerosis and in several of its major risk factors, including
    hypercholesterolemia, diabetes, obesity, hypertension. Levels serve as a
    predictor of adverse clinical outcome following vascular surgery, possibly as an
    indicator of thrombus formation (Sadler (1998) Annu Rev Biochem 67:395-424
    Blann et al. (1994) Eur J Vase Surg 8:10-15; Kessler et al. (1998) Diabetes
    Metab 24:327-36; Folsom et al. (1997) Circulation 96:1102-1108)
    platelet endothelial Signaling molecule in the migration of cells as part of the pathophysiology of
    cell adhesion vascular occulsive diseases such as atherosclerosis. Analysis of
    molecule endothelial/monocyte co-cultures indicates oxidative stress induces
    (PECAM-1) transendothelial migration of monocytes as a result of phosphorylation of
    PECAM-1 (Rattan et al. (1997) Am J Physiol 273:E453-61)
    antithrombin III ATIII is the sole blood component through which heparin exerts its anti-
    variant (AT3) coagulation effect. Deficiency in ATIII causes recurrent venous thrombosis
    and pulmonary embolism and can be inherited in autosomal dominant fashion
    (Hultin et al. (1988) Thromb Haemost 59:468-73; Lane et al. (1996) Blood Rev
    10:59-74)
    lipoprotein lipase Hydrolyses triglyceride in chylomicrons and therefore regulates metabolism of
    (LPL) circulating lipoproteins. Appears to have an atherogenic effect on the arterial
    wall due to its ability to alter the properties of LDL. Increased activity of LPL
    is found in atherosclerotic arteries when compared to normal. Expressed by
    macrophages in atherosclerotic lesions. Mutations in LPL responsible for
    familial hypercholesterolemia and premature atherosclerosis (Fisher et al.
    (1997) Atherosclerosis 135:145-159; Goldberg (1996) J Lipid Res 37:693-707;
    Gerdes et al. (1997) Circulation 96:733-740)
    alpha-2-macro- Foam cell formation—retains LDL cholesterol in the lipid core of
    globulin (A2M) atherosclerotic plaque (Llorente et al. (1998) Rev Esp Cardiol 51:633-641)
    apolipoprotein AI Participates in reverse cholesterol transport from tissues to the liver. Promotes
    (APOA1) cholesterol efflux from tissues and acts as a cofactor for lecithin cholesterol
    acyltransferase (LCAT). Mutations in ApoA1 and of ApoAI/CIII/AIV gene
    cluster assoc with atherosclerosis. Transgenic mice expressing high plasma
    APOAI levels are protected from fatty streak development with a high
    atherogenic diet (Gordon et al. (1989) Circulation 79:8-15; Rubin et al. (1991)
    Nature 353:265-7; Karathanasis et al. (1987) Proc Natl Acad Sci 84:7198-
    7202)
    apolipoprotein AII Major component of HDL. Appears to have an opposite effect to that of
    (APOA2) APOAI, though exact function unknown. APOAII may have ability to convert
    HDL from an anti- to a pro-inflammatory particle, with paraoxonase having a
    role in this transformation process. Plasma APOAII levels significantly
    associated with plasma free fatty acid levels. Transgenic mice expressing
    varying levels of APOAII show increased atherosclerotic lesions than wt when
    fed an atherogenic diet. Possible interaction between diet/genotype and
    atherogenic potential (Escola-Gil et al. (1998) J Lipid Res 39:457-462; Warden
    et al. (1993) Proc Natl Acad Sci 90:10886-10890)
    apolipoprotein B-100 Main apolipoprotein of chylomicrons and low density lipoproteins. Mutations in
    (APOB) APOB100 underly familial defective apolipoprotein B-100 in which patients
    suffer from premature atherosclerosis. Mutations result in defect in binding of
    LDL to LDL receptor, and accumulation of plasma LDL. High-expressing
    APOB transgenic mice exibit elevated VLDL-LDL cholesterol and atherogenic
    lesions (Callow et al. (1995) J Clin Invest 96:1639-1646; Brasaemle et al.(1997)
    J Biol Chem 272:9378-9387)
    lipoprotein apoCII Role in lipoprotein metabolism. Cofactor in the activity of lipoprotein lipase the
    (APOC2) enzyme that hydrolyzes triglycerides in plasma and transfers the fatty acids to
    tissues. Mutations in APOC2 responsible for hyperlipoproteinemia 1B, similar
    to lipoprotein lipase deficiency (Cox et al. (1978) N Engl J Med 299:1421-1424;
    Arimoto et al. (1998) J Lipid Res 39:143-151)
    pre-apolipoprotein Inhibits lipoprotein lipase and hepatic lipase, decreases uptake of lymph
    CIII (APOC3) chylomicrons by hepatic cells. APOA3 possibly delays breakdown of
    triglyceride rich particles. SstI RFLP in apoCIII is associated with plasma
    triglyceride and apoCIII levels and hyperlipidemic phenotypes (Henderson et
    al.(1987) Hum Genet 75:62-65)
    apolipoprotein apoC- APOC4 is a lipid-binding protein that has the potential to alter lipid metabolism.
    IV (APOC4) Human APOC4 transgenic mice are hypertriglyceridaemic compared to normal
    controls (Allan et al. (1996) J Lipid Res 37:1510-1518)
    macrophage Mediates binding, internalisation and processing of negatively-charged macro-
    scavenger receptor molecules. Implicated in the pathological deposition of cholesterol in arterial
    type I (MSR1) walls during atherogenesis (Han et al. (1998) Hum Mol Genet 7:1039-1046)
    Human antigen Acts as a scavenger receptor for oxidised LDL. Transient regulation under
    CD36 gene (CD36) control of M-CSF during monocyte-macrophage differentiation increases foam
    cell accumulation, Possible role in atherogenesis: increased M-CSF levels
    detected in atherosclerotic lesions in rabbits and humans (Huh et al. (1996)
    Blood 87:2020-2028; Aitman et al. (1999) Nat Genet 21:76-83)
    serum amyloid P Plasma glycoprotein expressed in atherosclerotic lesions. Interacts with
    component (SAP) lipoproteins in specific manner (Li et al. (1995) Arterioscler Thromb Vasc Biol
    15:252-257; Li et al. (1998) Biochem Biophys Res Commun 244:249-252)
    carboxyl ester lipase CEL gene expression increases in presence of oxidised and native LDL in
    gene (CEL) vitro. It is expressed in the vessel wall and in aortic extracts - may interact
    with cholesterol to modulate progression of atherosclerosis (Li et al. (1998)
    Biochem J 329:675-679)
    paraoxonase 1 Serum esterase exclusively associated with high-density lipoproteins; it might
    (PON1) confer protection against coronary artery disease by destroying pro-
    inflammatory oxidized lipids in oxidized low-density lipoproteins. PON1 gln192-
    to-arg polymorphism associated with coronary artery disease. Association
    between PON1 genetic variation and plasma LDL, HDL and non-HDL and
    apoB levels in genetically isolated Alberta Hutterite population. When fed on a
    high-fat, high-cholesterol diet, PON1-null mice were more susceptible to
    atherosclerosis than wild-type (Serrato et al. (1995) J Clin Invest 96:3005-3008;
    Boright et al. (1998) Atherosclerosis 139:131-136; Shih et al. (1998) Nature
    394:284-287)
    paraoxonase 2 Serum esterase exclusively associated with high-density lipoproteins; it might
    (PON2) confer protection against coronary artery disease by destroying pro-
    inflammatory oxidized lipids in oxidized low-density lipoproteins. Common
    polymorphism at codon 311 (cys-ser) in PON2 associated with CHD alone and
    synergistically with the 192 polymorphism in PON1 in Asian Indians.
    Association between genetic variation in PON2 and plasma cholesterol and
    apolipoprotein A1 in genetically isolated Alberta Hutterite population (Sanghera
    et al. (1998) Am J Hum Genet 62:36-44; Boright supra)
    paraoxonase 3 Serum esterase exclusively associated with high-density lipoproteins; it might
    (PON3) confer protection against coronary artery disease by destroying pro-
    inflammatory oxidized lipids in oxidized low-density lipoproteins. Other
    members PON2, 3 associated with CHD and cholesterol levels (Laplaud et al.
    (1998) Clin Chem Lab Med 36:431-441)
    perilipin (PLIN) Lipid storage droplets of steroidogenic cells are surrounded by perilipins, family
    of phosphorylated proteins encoded by a singlegene, detected in adipocytes and
    steroidogenic cells. Possible role in lipid metabolism (Brasaemle et al. (1997) J
    Biol Chem 272:9378-9387)
    Prostaglandin D2 Catalyses conversion of PGH2 to PGD2, a prostaglandin important in smooth
    synthase (PTGDS) muscle contraction/relaxation and potent inhibitor of platelet aggregation.
    Northern analysis shows strong specific expression in heart. Immunocyto-
    chemical localization to myocardial and atrio endocardial cells, and accumulates
    in end-stage atherosclerotic plaques. High plasma levels detected in severe
    angina patients (Eguchi et al. (1997) Proc Natl Acad Sci 94:14689-14694)
    Annexin Inhibits phospholipase A2 activity and the production of arachidonic acid, the
    II/lipocortinII(ANX2) precursor of the inflammatory mediators prostaglandins and leukotrienes.
    ANX2 is an important anti-inflammatory molecule that binds plasminogen and t-
    PA and is suspected of having a role in atherogenesis. Binding of plasminoger
    to ANX2 is specifically inhibited by the excess atherogenic Lp(a) (Hajjar et al.
    (1998) J Investig Med 46:364-369)
    Annexin I/lipocortin(ANX1) Inhibits phospholipase A2 activity and production of arachidonic acid, the
    precursor of the inflammatory mediators prostaglandins and leukotrienes.
    ANXI is an important anti-inflammatory molecule (Wallner et al. (1986) Nature
    320:77-81)
    Secreted protein, Extracellular glycoprotein secreted by endothelial cells which has a suspected
    acidic and rich in role in calcification of atherosclerotic plaques. Interacts with PDGF-B
    cysteine (SPARC) containing dimers and inhibits binding to its receptors. Expression of SPARC
    and PDGF is minimal in most adult tissues, but is enhanced following injury and
    advanced atherosclerotic lesions. Selective expression of SPARC causes
    rounding of adherent endothelial cells and influences extravasation of
    macromolecules (Raines et al. (1992) Proc Natl Acad Sci 89:1281-1285;
    Goldblum et al. (1994) Proc Natl Acad Sci 91:3448-3452)
  • From a total of 45,233 assembled gene sequences, 25 cDNAs were identified (SEQ ID NOs:1-25 of the Sequence Listing) that show strong association with the known atherosclerosis-associated genes. [0156]
  • Initially, the degree of association was measured by probability values using a cutoff p-value less than 0.00001. The sequences were further examined to ensure that the genes that passed the probability test had strong association with known atherosclerosis-associated genes. Details of the co-expression patterns for the known genes and co-expressed cDNAs are presented in Table 4. The entries in Table 4 are the negative log of the p-value (−log p) for the coexpression of the two genes. Table 5 summarizes the highly significant co-expression relationships between each cDNA, two marker genes and their functions. [0157]
  • V Atherosclerosis-Associated cDNAs [0158]
  • Using the co-expression analysis method, cDNAs comprising the polynucleotides of SEQ ID NOs:1-25 and their complements, were identified by their highly significant co-expression with known atherosclerosis-associated genes. [0159]
  • BLAST and other motif searches were performed for SEQ ID NOs:1-25 according to Example VII. SEQ ID NO:8 was determined to be full length and translated as shown in FIG. 1. [0160]
  • VI Transcript Imaging [0161]
  • Transcript images were performed for several of the cDNAs of the invention using the LIFESEQ GOLD database (July 02 release, Incyte Genomics). This process allowed assessment of the relative abundance of the expressed polynucleotides in all of the cDNA libraries, but those in the cardiovascular category are specifically emphasized. Criteria for transcript imaging can be selected from category, number of cDNAs per library, library description, disease indication, clinical relevance of sample, and the like. [0162]
  • All sequences and cDNA libraries in the LIFESEQ database have been categorized by system, organ/tissue and cell type. For each category, the number of libraries in which the sequence was expressed were counted and shown over the total number of libraries in that category. For each library, the number of cDNAs were counted and shown over the total number of cDNAs in that library. In some transcript images, all normalized or subtracted libraries, which have high copy number sequences removed prior to processing, and all mixed or pooled tissues, which are considered non-specific in that they contain more than one tissue type or more than one subject's tissue, can be excluded from the analysis. Treated and untreated cell lines and/or fetal tissue data can also be excluded where clinical relevance is emphasized. Conversely, fetal tissue can be emphasized wherever elucidation of inherited disorders or differentiation of particular adult or embryonic stem cells into tissues or organs such as heart, kidney, nerves or pancreas would be aided by removing clinical samples from the analysis. Transcript imaging can also be used to support data from other methodologies such as guilt-by-association and hybridization analyses. [0163]
  • The transcript images for SEQ ID NOs:3-6, 8,13, 15-20, and 22 are shown in Table 6. The first column shows library name; the second column, the number of cDNAs sequenced in that library; the third column, the description of the library; the fourth column, absolute abundance of the transcript in the library; and the fifth column, percentage abundance of the transcript in the library. In some cases, the normal library shows differential expression of the cDNA; in others, the induced or diseased library shows differential expression. For example, for SEQ ID NO:8, the endothelial cells treated with growth factors (VEGF and EF) show 2-8×higher expression than untreated endothelial cells in the same experiment. [0164]
  • These data confirm the differential expression of SEQ ID NOs:3-6, 8,13, 15-20, and 22 under conditions that correlate with disorders associated with atherosclerosis. VII Homology Searching for Atherosclerosis-Associated cDNAs and Polypeptides The polynucleotide sequences, SEQ ID NO:1-25, and polypeptide sequence, SEQ ID NO:26, were queried against databases derived from sources such as GenBank and SwissProt. These databases, which contain previously identified and annotated sequences, were searched for regions of similarity using BLAST (Altschul, supra). BLAST searched for matches and reported only those that satisfied the probability thresholds of 10-25 or less for nucleotide sequences and 10-8 or less for polypeptide sequences. [0165]
  • The polypeptide sequence was also analyzed for known motif patterns using MOTIFS, SPSCAN, BLIMPS, and HMM-based protocols. MOTIFS (Genetics Computer Group, Madison Wis.) searches polypeptide sequences for patterns that match those defined in the Prosite Dictionary of Protein Sites and Patterns (Bairoch, supra) and displays the patterns found and their corresponding literature abstracts. SPSCAN (Genetics Computer Group) searches for potential signal peptide sequences using a weighted matrix method (Nielsen et al. (1997) Protein Engineering 10:1-6). Hits with a score of 5 or greater were considered. BLIMPS uses a weighted matrix analysis algorithm to search for sequence similarity between the polypeptide sequences and those contained in BLOCKS, a database consisting of short amino acid segments, or blocks of 3-60 amino acids in length, compiled from the PROSITE database (Henikoff; supra; Bairoch, supra), and those in PRINTS, a protein fingerprint database based on non-redundant sequences obtained from sources such as SwissProt, GenBank, PIR, and NRL-3D (Attwood et al. (1997) J Chem Inf Comput Sci 37:417-424). For the purposes of the present invention, the BLIMPS searches reported matches with a cutoff score of 1000 or greater and a cutoff probability value of 1.0×10[0166] −3. HMM-based protocols were based on a probabilistic approach and searched for consensus primary structures of gene families in the protein sequences (Eddy, supra; Sonnhammer, supra). More than 500 known protein families with cutoff scores ranging from 10 to 50 bits were selected for use in this invention.
  • VIII Hybridization Technologies: Selection of Sequences, Microarray Preparation and Use [0167]
  • SEQ ID NO:1-25 are represented among the template sequences in the LIFESEQ GOLD database (Incyte Genomics). Several of these sequences, specifically SEQ ID NOs:1, 2, 10, 12, 18, and 32-34 have been used on microarrays in experiments investigating differential gene expression in cardiovascular samples. Table 7 presents the results of these experiments; results were significant if the log2 Cy/Cy5 ratio exceeded ±1.00 in either the normal or the induced or disease state. [0168]
  • Exemplary Experimental Materials and Protocols [0169]
  • Activation of the vascular endothelium is considered to be a central event in a wide range of both physiological and pathophysiological processes, such as vascular tone regulation, coagulation and thrombosis, atherosclerosis, and inflammation. [0170]
  • HAEC, human aortic endothelial cells, are primary cells derived from the endothelium of a human aorta. They have been used as an experimental model for investigating the role of the endothelium in human vascular biology in vitro. HAECs were grown to 85% confluency, split into two samples, one of which was then treated with growth factors, LDL, cytokines, O2, and the like for variable time periods. [0171]
  • ECV304, HUAEC, and HUVEC are endothelial cell lines derived from the endothelium of the human umbilical artery or vein. This cell model has been extensively used to study the functional biology of human endothelial cells. These cells were also grown to about 85% confluency, split into samples, one of which was then treated with growth factors, LDL, cytokines, O2, and the like for variable time periods. [0172]
  • The experimental treatments and time of exposure are shown by the tissue description in Table 7. [0173]
  • Exemplary Activators and Inducers [0174]
  • TNF-α is a pleiotropic cytokine that is known to play a central role in the mediation of inflammatory responses through activation of multiple signal transduction pathways. TNF-α is produced by activated lymphocytes, macrophages, and other white blood cells, and is known to activate endothelial cells. Monitoring the endothelial cells' response to TNF-α at the level of the mRNA expression can provide information necessary for better understanding of both TNF-α signaling pathways and endothelial cell biology. [0175]
  • PMA is a broad activator of the protein kinase C-dependent pathways. lonomycin is a calcium ionophore that permits the entry of calcium in the cell, hence increasing the cytosolic calcium concentration. The combination of PMA and ionomycin activates two of the major signaling pathways used by mammalian cells to interact with their environment. In T cells, the combination of PMA and ionomycin mimics the type of secondary signaling events elicited during optimal B cell activation. [0176]
  • Microarrays [0177]
  • The HUMAN GENOME GEM series 1-5 microarrays (Incyte Genomics) contain 45,320 array elements which represent 22,632 annotated clusters and 22,688 unannotated clusters. For the UNIGEM series microarrays (Incyte Genomics), Incyte clones were mapped to non-redundant Unigene clusters (Unigene database (build 46), NCBI; Shuler (1997) J Mol Med 75:694-698), and the 5′ clone with the strongest BLAST alignment (at least 90% identity and 100 bp overlap) was chosen, verified, and used in the construction of the microarray. The UNIGEM V 2.0 microarray (Incyte Genomics) contains 8,502 array elements which represent 8,372 annotated genes and 130 unannotated clusters. [0178]
  • To construct microarrays, cDNAs were amplified from bacterial cells using primers complementary to vector sequences flanking the cDNA insert. Thirty cycles of PCR increased the initial quantity of cDNAs from 1-2 ng to a final quantity greater than 5 μg. Amplified cDNAs were then purified using SEPHACRYL-400 columns (APB). Purified cDNAs were immobilized on polymer-coated glass slides. Glass microscope slides (Corning, Corning N.Y.) were cleaned by ultrasound in 0.1% SDS and acetone, with extensive distilled water washes between and after treatments. Glass slides were etched in 4% hydrofluoric acid (VWR Scientific Products, West Chester Pa.), washed thoroughly in distilled water, and coated with 0.05% aminopropyl silane (Sigma-Aldrich) in 95% ethanol. Coated slides were cured in a 110° C. oven. cDNAs were applied to the coated glass substrate using a procedure described in U.S. Pat. No. 5,807,522. One microliter of the cDNA at an average concentration of 100 ng/μl was loaded into the open capillary printing element by a high-speed robotic apparatus which then deposited about 5 nl of cDNA per slide. [0179]
  • Microarrays were UV-crosslinked using a STRATALINKER UV-crosslinker (Stratagene), and then washed at room temperature once in 0.2% SDS and three times in distilled water. Non-specific binding sites were blocked by incubation of microarrays in 0.2% casein in phosphate buffered saline (Tropix, Bedford Mass.) for 30 minutes at 60° C. followed by washes in 0.2% SDS and distilled water as before. [0180]
  • Isolation and Labeling of Sample cDNAs [0181]
  • Cells were harvested and lysed in 1 ml of TRIZOL reagent (5×10[0182] 6 cells/ml; Invitrogen). The lysates were vortexed thoroughly and incubated at room temperature for 2-3 minutes and extracted with 0.5 ml chloroform. The extract was mixed, incubated at room temperature for 5 minutes, and centrifuged at 16,000×g for 15 minutes at 4° C. The aqueous layer was collected, and an equal volume of isopropanol was added. Samples were mixed, incubated at room temperature for 10 minutes, and centrifuged at 16,000×g for 20 minutes at 4° C. The supernatant was removed, and the RNA pellet was washed with 1 ml of 70% ethanol, centrifuged at 16,000×g at 4° C., and resuspended in RNAse-free water. The concentration of the RNA was determined by measuring the optical density at 260 nm.
  • Poly(A) RNA was prepared using an OLIGOTEX mRNA kit (Qiagen) with the following modifications: OLIGOTEX beads were washed in tubes rather than spin columns, resuspended in elution buffer, and then loaded onto spin columns to recover the mRNA. To obtain maximum yield, the mRNA was eluted twice. [0183]
  • Each poly(A) RNA sample was reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/μl oligo-d(T) primer (21mer), 1×first strand buffer, 0.03 units/ul RNAse inhibitor, 500 uM dATP, 500 uM dGTP, 500 uM dTTP, 40 uM dCTP, and 40 uM either dCTP-Cy3 or dCTP-Cy5 (APB). The reverse transcription reaction was performed in a 25 ml volume containing 200 ng poly(A) RNA using the GEMBRIGHT kit (Incyte Genomics). Specific control poly(A) RNAs (YCFRO6, YCFR45, YCFR67, YCFR85, YCFR43, YCFR22, YCFR23, YCFR25, YCFR44, YCFR26) were synthesized by in vitro transcription from non-coding yeast genomic DNA (W. Lei, unpublished). As quantitative controls, control mRNAs (YCFRO6, YCFR45, YCFR67, and YCFR85) at 0.002 ng, 0.02 ng, 0.2 ng, and 2 ng were diluted into reverse transcription reaction at ratios of 1:100,000, 1:10,000, 1:1000, 1:100 (w/w) to sample mRNA, respectively. To sample differential expression patterns, control mRNAs (YCFR43, YCFR22, YCFR23, YCFR25, YCFR44, YCFR26) were diluted into reverse transcription reaction at ratios of 1:3, 3:1, 1:10, 10:1, 1:25, 25:1 (w/w) to sample mRNA. Reactions were incubated at 37° C. for 2 hr, treated with 2.5 ml of 0.5M sodium hydroxide, and incubated for 20 minutes at 85° C. to the stop the reaction and degrade the RNA. [0184]
  • cDNAs were purified using two successive CHROMA SPIN 30 gel filtration spin columns (Clontech). Cy3- and Cy5-labeled reaction samples were combined as described below and ethanol precipitated using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100% ethanol. The cDNAs were then dried to completion using a SpeedVAC system (Savant Instruments, Holbrook N.Y.) and resuspended in 14 [0185] μl 5×SSC, 0.2% SDS.
  • Hybridization and Detection [0186]
  • Hybridization reactions contained 9 μl of sample mixture containing 0.2 μg each of Cy3 and Cy5 labeled cDNA synthesis products in 5×SSC, 0.2% SDS hybridization buffer. The mixture was heated to 65° C. for 5 minutes and was aliquoted onto the microarray surface and covered with an 1.8 cm[0187] 2 coverslip. The microarrays were transferred to a waterproof chamber having a cavity just slightly larger than a microscope slide. The chamber was kept at 100% humidity internally by the addition of 140 μl of 5×SSC in a corner of the chamber. The chamber containing the microarrays was incubated for about 6.5 hours at 60° C. The microarrays were washed for 10 min at 45° C. in low stringency wash buffer (1×SSC, 0.1% SDS), three times for 10 minutes each at 45° C. in high stringency wash buffer (0.1×SSC), and dried.
  • Reporter-labeled hybridization complexes were detected with a microscope equipped with an Innova 70 mixed gas 10 W laser (Coherent, Santa Clara Calif.) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of Cy5. The excitation laser light was focused on the microarray using a 20×microscope objective (Nikon, Melville N.Y.). The slide containing the microarray was placed on a computer-controlled X-Y stage on the microscope and raster-scanned past the objective. The 1.8 cm×1.8 cm microarray used in the present example was scanned with a resolution of 20 micrometers. [0188]
  • In two separate scans, the mixed gas multiline laser excited the two fluorophores sequentially. Emitted light was split, based on wavelength, into two photomultiplier tube detectors (PMT R1477; Hamamatsu Photonics Systems, Bridgewater N.J.) corresponding to the two fluorophores. Appropriate filters positioned between the microarray and the photomultiplier tubes were used to filter the signals. The emission maxima of the fluorophores used were 565 nm for Cy3 and 650 nm for Cy5. Each microarray was typically scanned twice, one scan per fluorophore using the appropriate filters at the laser source, although the apparatus was capable of recording the spectra from both fluorophores simultaneously. [0189]
  • The sensitivity of the scans was calibrated using the signal intensity generated by a cDNA control species. Samples of the calibrating cDNA were separately labeled with the two fluorophores and identical amounts of each were added to the hybridization mixture. A specific location on the microarray contained a complementary DNA sequence, allowing the intensity of the signal at that location to be correlated with a weight ratio of hybridizing species of 1:100,000. [0190]
  • The output of the photomultiplier tube was digitized using a 12-bit RTI-835H analog-to-digital (A/D) conversion board (Analog Devices, Norwood, Mass.) installed in an IBM-compatible PC computer. The digitized data were displayed as an image where the signal intensity was mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal). The data was also analyzed quantitatively. Where two different fluorophores were excited and measured simultaneously, the data were first corrected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore's emission spectrum. [0191]
  • A grid was superimposed over the fluorescence signal image such that the signal from each spot was centered in each element of the grid. The fluorescence signal within each element was then integrated to obtain a numerical value corresponding to the average intensity of the signal. The software used for signal analysis was the GEMTOOLS gene expression analysis program (Incyte Genomics). Significance was defined as signal to background ratio exceeding 2×and area hybridization exceeding 40%. [0192]
  • IX Further Characterization of Differentially Expressed cDNAs and Proteins [0193]
  • Clones were aligned against the LIFESEQ Gold 5.1 database (Incyte Genomics) and an Incyte template and its sequence variants were chosen for each clone. The template and variant sequences were aligned against the GenBank nucleotide sequence databases using BLASTn (vers. 2.0, NCBI) to acquire annotation. The template and variant sequences were translated into amino acid sequences which were aligned against GenPept and other protein databases using BLASTp (vers. 2.0, NCBI) to acquire annotation and characterization, i.e., structural motifs. Table 3 shows the GenBank annotations (where available) for SEQ ID NOs:1-25 of this invention as produced by BLAST analysis. [0194]
  • Percent sequence identity can be determined electronically for two or more amino acid or nucleic acid sequences using the MEGALIGN program, a component of LASERGENE software (DNASTAR). The percent identity between two amino acid sequences is calculated by dividing the length of sequence A, minus the number of gap residues in sequence A, minus the number of gap residues in sequence B, into the sum of the residue matches between sequence A and sequence B, times one hundred. Gaps of low or of no homology between the two amino acid sequences are not included in determining percentage identity. [0195]
  • Sequences with conserved protein motifs may be searched using the BLOCKS search program. This program analyses sequence information contained in the Swiss-Prot and PROSITE databases and is useful for determining the classification of uncharacterized proteins translated from genomic or cDNA sequences (Bairoch, supra; Attwood, supra). PROSITE database is a useful source for identifying functional or structural domains that are not detected using motifs due to extreme sequence divergence. Using weight matrices, these domains are calibrated against the SWISS-PROT database to obtain a measure of the chance distribution of the matches. [0196]
  • The PRINTS database can be searched using the BLIMPS search program to obtain protein family “fingerprints”. The PRINTS database complements the PROSITE database by exploiting groups of conserved motifs within sequence alignments to build characteristic signatures of different protein families. For both BLOCKS and PRINTS analyses, the cutoff scores for local similarity were: [0197]
  • >1300=strong, 1000-1300=suggestive; for global similarity were: p<exp−3; and for strength (degree of correlation) were: >1300=strong, 1000-1300=weak. [0198]
  • X Other Hybridization Technologies and Analyses [0199]
  • Other hybridization technologies utilize a variety of substrates such as nylon membranes, capillary tubes, etc. Arranging cDNAs on polymer coated slides is described in Example V; sample cDNA preparation and hybridization and analysis using polymer coated slides is described in examples VI and VII, respectively. [0200]
  • The cDNAs are applied to a membrane substrate by one of the following methods. A mixture of cDNAs is fractionated by gel electrophoresis and transferred to a nylon membrane by capillary transfer. Alternatively, the cDNAs are individually ligated to a vector and inserted into bacterial host cells to form a library. The cDNAs are then arranged on a substrate by one of the following methods. In the first method, bacterial cells containing individual clones are robotically picked and arranged on a nylon membrane. The membrane is placed on LB agar containing selective agent (carbenicillin, kanamycin, ampicillin, or chloramphenicol depending on the vector used) and incubated at 37° C. for 16 hr. The membrane is removed from the agar and consecutively placed colony side up in 10% SDS, denaturing solution (1.5 M NaCl, 0.5 M NaOH), neutralizing solution (1.5 M NaCl, 1 M Tris, pH 8.0), and twice in 2×SSC for 10 min each. The membrane is then UV irradiated in a STRATALINKER UV-crosslinker (Stratagene). [0201]
  • In the second method, cDNAs are amplified from bacterial vectors by thirty cycles of PCR using primers complementary to vector sequences flanking the insert. PCR amplification increases a starting concentration of 1-2 ng nucleic acid to a final quantity greater than 5 μg. Amplified nucleic acids from about 400 bp to about 5000 bp in length are purified using SEPHACRYL-400 beads (APB). Purified nucleic acids are arranged on a nylon membrane manually or using a dot/slot blotting manifold and suction device and are immobilized by denaturation, neutralization, and UV irradiation as described above. [0202]
  • Hybridization probes derived from cDNAs of the Sequence Listing are employed for screening cDNAs, mRNAs, or genomic DNA in membrane-based hybridizations. Probes are prepared by diluting the cDNAs to a concentration of 40-50 ng in 45 IL TE buffer, denaturing by heating to 100° C. for five min and briefly centrifuging. The denatured cDNA is then added to a REDIPRIME tube (APB), gently mixed until blue color is evenly distributed, and briefly centrifuged. Five microliters of [[0203] 32P]dCTP is added to the tube, and the contents are incubated at 37° C. for 10 min. The labeling reaction is stopped by adding 5 μl of 0.2M EDTA, and probe is purified from unincorporated nucleotides using a PROBEQUANT G-50 microcolumn (APB). The purified probe is heated to 100° C. for five min and then snap cooled for two min on ice.
  • Membranes are pre-hybridized in hybridization solution containing 1% Sarkosyl and Ix high phosphate buffer (0.5 M NaCl, 0.1 M Na[0204] 2HPO4, 5 mM EDTA, pH 7) at 55° C. for two hr. The probe, diluted in 15 ml fresh hybridization solution, is then added to the membrane. The membrane is hybridized with the probe at 55° C. for 16 hr. Following hybridization, the membrane is washed for 15 min at 25° C. in imM Tris (pH 8.0), 1% Sarkosyl, and four times for 15 min each at 25° C. in 1 mM Tris (pH 8.0). To detect hybridization complexes, XOMAT-AR film (Eastman Kodak, Rochester N.Y.) is exposed to the membrane overnight at −70° C., developed, and examined.
  • XI Expression of the Encoded Protein [0205]
  • Expression and purification of a protein encoded by a cDNA of the invention is achieved using bacterial or virus-based expression systems. For expression in bacteria, cDNA is subcloned into a vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription. Examples of such promoters include, but are not limited to, the trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element. Recombinant vectors are transformed into bacterial hosts, such as BL21(DE3). Antibiotic resistant bacteria express the protein upon induction with IPTG. Expression in eukaryotic cells is achieved by infecting [0206] Spodoptera frugiperda (Sf9) insect cells with recombinant baculovirus, Autographica californica nuclear polyhedrosis virus. The polyhedrin gene of baculovirus is replaced with the cDNA by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of transcription.
  • For ease of purification, the protein is synthesized as a fusion protein with glutathione-S-transferase (GST; APB) or a similar alternative such as FLAG. The fusion protein is purified on immobilized glutathione under conditions that maintain protein activity and antigenicity. After purification, the GST moiety is proteolytically cleaved from the protein with thrombin. A fusion protein with FLAG, an 8-amino acid peptide, is purified using commercially available monoclonal and polyclonal anti-FLAG antibodies (Eastman Kodak, Rochester N.Y.). [0207]
  • XII Production of Antibodies [0208]
  • A denatured protein from a reverse phase HPLC separation is obtained in quantities up to 75 mg. This denatured protein is used to immunize mice or rabbits following standard protocols. About 100 μg is used to immunize a mouse, while up to 1 mg is used to immunize a rabbit. The denatured protein is radioiodinated and incubated with murine B-cell hybridomas to screen for monoclonal antibodies. About 20 mg of protein is sufficient for labeling and screening several thousand clones. [0209]
  • In another approach, the amino acid sequence translated from a cDNA of the invention is analyzed using PROTEAN software (DNASTAR) to determine antigenic determinants of the protein. The optimal sequences for immunization are usually at the C-terminus, the N-terminus, and those intervening, hydrophilic regions of the protein that are likely to be exposed to the external environment when the protein is in its natural conformation. Typically, oligopeptides about 15 residues in length are synthesized using an 431 peptide synthesizer (ABI) using Fmoc-chemistry and then coupled to keyhole limpet hemocyanin (KLH; Sigma-Aldrich) by reaction with M-maleimidobenzoyl-N-hydroxysuccinimide ester. If necessary, a cysteine may be introduced at the N-terminus of the peptide to permit coupling to KLH. Rabbits are immunized with the oligopeptide-KLH complex in complete Freund's adjuvant. The resulting antisera are tested for antipeptide activity by binding the peptide to plastic, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radioiodinated goat anti-rabbit IgG. [0210]
  • Hybridomas are prepared and screened using standard techniques. Hybridomas of interest are detected by screening with radioiodinated protein to identify those fusions producing a monoclonal antibody specific for the protein. In a typical protocol, wells of 96 well plates (FAST, Becton-Dickinson, Palo Alto Calif.) are coated with affinity-purified, specific rabbit-anti-mouse (or suitable anti-species Ig) antibodies at 10 mg/ml. The coated wells are blocked with 1% BSA and washed and exposed to supernatants from hybridomas. After incubation, the wells are exposed to radiolabeled protein at 1 mg/ml. Clones producing antibodies bind a quantity of labeled protein that is detectable above background. [0211]
  • Such clones are expanded and subjected to 2 cycles of cloning at 1 cell/3 wells. Cloned hybridomas are injected into pristane-treated mice to produce ascites, and monoclonal antibody is purified from the ascitic fluid by affinity chromatography on protein A (APB). Monoclonal antibodies with affinities of at least 10[0212] 8 M−, preferably 109 to 1010 M−1 or stronger, are made by procedures well known in the art.
  • XIII Purification of Naturally Occurring Protein Using Antibodies [0213]
  • Naturally occurring or recombinant protein is purified by immunoaffinity chromatography using antibodies specific for the protein. An immunoaffinity column is constructed by covalently coupling the antibody to CNBr-activated SEPHAROSE resin (APB). Media containing the protein is passed over the immunoaffinity column, and the column is washed using high ionic strength buffers in the presence of detergent to allow preferential absorbance of the protein. After coupling, the protein is eluted from the column using a buffer of pH 2-3 or a high concentration of urea or thiocyanate ion to disrupt antibody/protein binding, and the protein is collected. [0214]
  • XIV Screening for Molecules That Specifically Bind the cDNA or Protein [0215]
  • The cDNA or fragments thereof and the protein or portions thereof are labeled with [0216] 32P-dCTP, Cy3-dCTP, Cy5-dCTP (APB), or BIODIPY or FITC (Molecular Probes), respectively. Candidate molecules or compounds previously arranged on a substrate are incubated in the presence of labeled nucleic or amino acid. After incubation under conditions for either a cDNA or a protein, the substrate is washed, and any position on the substrate retaining label, which indicates specific binding or complex formation, is assayed. The binding molecule is identified by its arrayed position on the substrate. Data obtained using different concentrations of the nucleic acid or protein are used to calculate affinity between the labeled nucleic acid or protein and the bound molecule. High throughput screening using very small assay volumes and very small amounts of test compound is fully described in U.S. Pat. No. 5,876,946.
  • All patents and publications mentioned in the specification are incorporated herein by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the field of molecular biology or related fields are intended to be within the scope of the following claims. [0217]
    TABLE 4
    Co-expression of 25 cDNAs with known atherosclerosis genes (- log p).
    SEQ ID CNN1 COL1A1 COL1A2 COL6A1 COL5A2 COL6A2 COL6A3 COL3A1 COL5A1 MGP CTSK FGB VWF
    1 0 1 0 0 0 0 0 0 0 0 0 4 1
    2 1 1 3 2 2 2 4 3 4 5 3 1 6
    3 0 1 0 1 1 1 0 0 1 1 1 4 0
    4 1 0 1 1 1 0 1 1 1 0 1 7 0
    5 5 9 12 13 3 14 11 12 4 10 13 1 3
    6 6 4 7 9 4 13 5 6 8 14 6 1 12
    7 5 4 5 5 2 7 5 6 5 10 1 2 1
    8 1 1 0 2 1 1 0 1 0 1 0 1 1
    9 1 1 4 2 1 6 4 4 5 6 1 0 10
    10 3 4 3 3 3 4 4 3 1 4 1 0 5
    11 1 1 1 1 1 1 1 0 1 1 2 1 1
    12 11 4 7 9 2 9 11 10 7 11 3 0 5
    13 2 6 5 6 9 8 4 6 7 1 3 1 1
    14 0 0 0 0 0 0 0 1 0 1 1 4 1
    15 17 7 13 17 2 25 12 11 6 21 6 3 7
    16 13 22 27 27 15 33 24 34 15 20 18 0 8
    17 2 1 3 3 0 2 6 3 1 9 1 0 7
    18 6 5 6 7 3 7 10 6 2 14 5 0 5
    19 0 1 1 2 1 1 0 1 0 0 1 7 0
    20 8 7 8 12 3 9 11 10 3 11 6 1 8
    21 0 2 4 2 4 2 5 5 3 2 1 0 4
    22 1 1 0 1 3 1 2 1 1 1 1 4 1
    23 1 1 0 1 0 1 0 1 0 0 0 7 0
    24 0 1 2 0 0 0 0 1 0 1 0 8 1
    25 48 11 17 24 3 26 18 17 13 20 6 1 8
    SEQ ID PECAM1 AT3 LPL A2M APOA1 APOA2 APOB APOC2 APOC3 APOC4 MSR1 CD36 SAP CEL
    1 0 5 0 1 3 4 2 2 5 4 0 0 6 0
    2 7 0 1 4 0 1 0 0 0 0 2 3 0 1
    3 0 8 1 1 5 4 3 3 5 7 0 0 8 1
    4 0 4 1 2 4 5 4 4 6 2 0 1 3 0
    5 2 0 4 9 1 0 0 1 0 0 4 2 0 2
    6 6 1 5 7 0 1 1 0 1 0 7 3 0 2
    7 4 3 3 8 1 1 2 0 2 1 2 7 2 0
    8 0 0 3 3 0 0 0 2 0 0 1 1 1 0
    9 8 0 5 6 1 1 1 2 0 0 5 6 0 1
    10 3 0 4 4 0 0 0 0 0 0 9 5 0 0
    11 1 2 1 0 3 1 2 2 1 0 0 2 3 6
    12 2 1 3 6 2 0 1 0 0 0 4 3 0 3
    13 0 0 1 1 3 2 1 2 1 0 1 2 1 3
    14 0 5 1 1 4 5 5 5 3 1 0 0 2 1
    15 5 1 1 13 1 1 1 0 1 0 10 1 1 0
    16 7 0 3 12 1 0 0 1 1 0 9 5 0 1
    17 5 0 6 7 0 0 0 1 0 0 5 9 0 1
    18 4 0 6 10 1 0 1 0 0 0 8 5 1 1
    19 1 8 1 3 5 8 4 6 8 9 1 1 11 0
    20 2 0 12 10 1 1 0 1 0 0 9 14 0 0
    21 4 0 8 5 0 1 0 1 1 0 5 11 1 1
    22 4 9 0 2 6 8 7 6 4 3 1 1 7 0
    23 0 6 0 1 6 7 7 4 8 1 0 1 7 1
    24 0 7 0 2 8 10 10 7 8 4 0 0 5 1
    25 2 0 2 20 1 0 3 0 0 0 10 4 0 1
    SEQ ID PON1 PON2 PON3 PLIN PTGDS ANX2 ANX1 SPARC SM22
    1 5 1 2 0 0 2 0 1 0
    2 1 6 1 1 2 3 4 8 3
    3 5 2 2 0 0 1 0 1 0
    4 2 0 1 0 0 1 0 1 1
    5 1 1 1 6 4 6 3 9 9
    6 1 4 0 11 7 6 4 11 14
    7 1 4 5 2 1 11 7 7 8
    8 1 5 0 1 23 1 1 4 2
    9 0 7 1 6 3 3 2 7 4
    10 0 1 0 15 2 2 1 2 4
    11 6 2 7 4 0 0 2 1 0
    12 1 3 1 3 3 6 3 7 9
    13 2 3 2 2 1 1 3 4 2
    14 2 2 1 1 0 0 0 1 0
    15 0 2 1 1 12 7 9 14 27
    16 1 2 2 7 6 12 9 22 20
    17 0 1 0 6 2 3 1 6 3
    18 1 2 3 8 6 3 10 6 11
    19 5 1 2 0 1 0 1 0 1
    20 0 0 0 19 4 4 2 7 10
    21 0 1 0 8 0 2 2 3 3
    22 4 2 2 1 2 1 3 1 1
    23 4 1 3 1 0 0 0 1 0
    24 3 1 1 0 2 1 1 1 0
    25 1 1 1 3 11 9 6 9 30
  • [0218]
    TABLE 5
    Summary of coexpressed genes and their function in atherosclerosis-associated disorders
    SEQ ID Pvalue Gene 1 Gene function from Table 3 Pvalue Gene 2 Gene function from Table 3
    1 6 SAP associated with amyloid P 5 APOC3 associated with plasma
    in lesions triglyceride and hyperlipidemia
    2 8 SPARC calcification of plaques 7 PECAM- implicated in migration of
    cells under O2 stress
    3 8 AT3 deficiency causes recurrent 8 SAP associated with amyloid P in
    venous thrombosis lesions
    4 7 FGB fibrin deposition in plaque 6 APOC3 apolipoprotein CIII
    5 14 COL6A2 promoting platelet aggregation 12 CTSK present in advanced plaques
    6 14 SM22 downregulated during 14 MGP calcificaton associated with
    atherogenesis high expression
    7 11 ANX2 role in atherogenesis 10 MGP calcificaton associated with
    high expression
    8 23 PTDGS accumulates in end-stage 15 AGT associated with higher total
    atherosclerotic plaques cholesterol levels
    9 10 VWF Increased levels found in 8 PECAM- implicated in migration of
    atherosclerosis cells under O2 stress
    10 15 PLIN functions in lipid metabolism 9 MSR1 deposits cholesterol during
    atherogenesis
    11 7 PON3 associated with coronary heart 6 CEL implicated in the progression
    disease and cholesterol levels of atherosclerosis
    12 11 COL6A3 promoting platelet aggregation 11 MGP calcificaton associated with
    high expression
    13 9 COL5A2 promoting platelet aggregation 8 COL6A2 promoting platelet aggregation
    14 5 APOA/B accumulation of plasma LDL in 5 AT3 deficiency causes recurrent
    atherogenesis venous thrombosis
    15 27 SM22 downregulated during 25 COL6A2 promoting platelet aggregation
    atherogenesis
    16 34 COL3A1 promoting platelet aggregation 22 SPARC calcification of plaques
    17 9 MGP calcificaton associated with 9 CD36 receptor for oxidised LDL
    high expression
    18 11 SM22 downregulated during 10 A2M retains LDL in core of plaque
    atherogenesis
    19 11 SAP associated with amyloid P in 9 APOC4 alters lipid metabolism toward
    lesions hypertriglyceridaemia
    20 19 PLIN functions in lipid metabolism 14 CD36 receptor for oxidised LDL
    21 11 CD36 receptor for oxidised LDL 8 PLIN functions in lipid metabolism
    22 9 AT3 deficiency causes recurrent 8 APOA2 associated with increased
    venous thrombosis atherosclerotic lesions
    23 8 APOC3 associated with plasma 7 SAP associated with amyloid P in
    triglyceride and hyperlipidemia lesions
    24 10 APOA/B accumulation of plasma LDL in 8 FGB fibrin deposition in plaque
    atherogenesis
    25 48 CNN1 modulation of SMC, a feature 26 COL6A2 promoting platelet aggregation
    of atherosclerosis
  • [0219]
    TABLE 6
    Cardiovascular transcript images for the coexpressed cDNAs
    SEQ ID Library cDNAs Description of Sample Abundance % Abundance
    3 SMCRUNT01 3472 renal vein, smooth muscle cells, 57M, Untx 1 0.0288
    ENDVUNT01 5215 microvascular, dermal, endothelial cells, 22F, Untx 1 0.0192
    HEAANOT01 12578 heart, coronary artery, CAD, 46M 1 0.0080
    4 MONOTXN05 2709 periph blood, monocytes, 42F, t/IL-10, LPS, NORM 1 0.0369
    MONOTXT02 3554 periph blood, monocytes, 42F, t/IL-10, LPS 1 0.0281
    MCLRUNT01 6149 periph blood mononuclear cells, 60M, untreated 1 0.0163
    5 ARTANOT06 6311 aorta, adventitia, 48M 2 0.0317
    HEAONOT02 3482 heart, aorta, 10M 1 0.0287
    HEAONOT04 4002 heart, aorta, 12F 1 0.0250
    ARTANOT07 5716 aorta, adventitia, 65F 1 0.0175
    6 HEAONOE01 3639 heart, aorta, 39M, 5RP 10 0.2748
    HEAONOT03 3720 heart, aorta, aw/cerebral agenesis, 27F 3 0.0806
    HEAPNOT01 3502 heart, coronary artery, plaque, pool 2 0.0571
    8 ENDVTXT01 1882 microvascular, dermal, endothelial cells, 22F, t/bFGF, EF 3 0.1594
    ENDVTXT02 1876 microvascular, dermal, endothelial cells, 22F, t/VEGE, EF 1 0.0533
    ENDVUNT01 5215 microvascular, dermal, endothelial cells, 22F, Untx 1 0.0192
    13 SMCCNOS01 3494 coronary artery, smooth muscle cells, 3M, t/TNF, IL-1, SUB 1 0.0286
    SMCANOT01 7327 aortic smooth muscle line, M 1 0.0136
    15 SMCRTXT01 3453 renal vein, smooth muscle cells, 57M t/TNF, IL1 2 0.0579
    SMCRUNT01 3472 renal vein, smooth muscle cells, 57M, Untx 1 0.0288
    16 SMCCNOT01 4266 coronary artery, smooth muscle cells, 3M 5 0.1172
    SMCCNOT02 3980 coronary artery, smooth muscle cells, 3M, t/TNF, IL-1 2 0.0503
    SMCCNOS01 3494 coronary artery, smooth muscle cells, 3M, t/TNF, IL-1, SUB 1 0.0286
    17 ENDIUNT01 3582 iliac artery, endothelial cells, F, control, untreated 26 0.7259
    ENDITXT01 3464 iliac artery, endothelial cells, F, t/1% oxygen 24 hr 21 0.6062
    ENDINOT02 3208 iliac artery, endothelial cells, F, t/TNF, IL-1 20 hr 10 0.3117
    18 ENDVTXT01 1882 microvascular, dermal, endothelial cells, 22F, t/bFGF, EF 1 0.0531
    ENDVNOT01 4955 microvascular, dermal, endothelial cells, 18F, untreated 1 0.0202
    19 ENDATXP01 1877 aorta, endothelial cells, t/TNF, TIGR 1 0.0533
    ENDVTXT02 1876 microvascular, dermal, endothelial cells, 22F, t/VEGF, EF 1 0.0533
    ENDVTXT01 1882 microvascular, dermal, endothelial cells, 22F, t/bFGF, EF 1 0.0531
    ENDVNOT01 4955 microvascular, dermal, endothelial cells, 18F, untreated 1 0.0202
    20 HEAANOT01 12578 heart, coronary artery, CAD, 46M 13 0.1034
    HEAONOT05 3959 heart, aorta, 17F 3 0.0758
    HEAONOT04 4002 heart, aorta, 12F 1 0.0250
    22 ENDITXT01 3464 iliac artery, endothelial cells, F, t/1% oxygen 24 hr 1 0.0289
    HEAONOE01 3639 heart, aorta, 39M, 5RP 1 0.0275
    HEAANOT01 12578 heart, coronary artery, CAD, 46M 1 0.0080
  • [0220]
    TABLE 7
    Microarray data from cardiovascular experiments
    SEQ ID GEM Log2 (Cy5/Cy3) Cy3 Sample Cy5 Sample
    1 LG1 −1.01 ECV304 Line, Untx ECV304 Line, t/PMA + Iono
    2 HG1 −1.03 HUVEC Cells, Untx, HUVEC Cells, t/TNFa 10 ng/mL
    Nrml 1 hr, Nrml
    2 HG1 −1.03 HUAEC Cells, Untx, HUAEC Cells, t/TNFa 10 ng/mL
    Nrml 24 hr, Nrml
    2 HG1 −1.03 HUVEC Cells, Untx, HUVEC Cells, t/IL1b + TNFa
    Nrml 10 ng/mL, 10 ng/mL 2 4hr, 4 hr, Nrml
    2 HG1 −1.07 HUVEC Cells, Untx, HUVEC Cells, t/PMA + TNFa
    Nrml, 24 hr 10 nM, 10 ng/mL 24 hr, 1 hr, Nrml
    2 HG1 −1.07 HUVEC Cells, Untx, HUVEC Cells, t/TNFa 10 ng/mL
    Nrml 48 hr, Nrml
    2 HG1 −1.10 HUVEC Cells, Untx HUVEC Cells, t/IL4 10 ng/mL 24 hr
    2 HG1 −1.10 HUVEC Cells, Untx, HUVEC Cells, t/TNFa 10 ng/mL
    Nrml 2 hr, Nrml
    2 HG1 −1.12 HUVEC Cells, Untx, HUVEC Cells, t/IL4 + TNFa
    48 hr 10 ng/mL, 10 ng/mL 24 hr, 1 hr
    2 HG1 −1.12 HUVEC Cells, Untx HUVEC Cells, t/IL10 + TNFa
    10 ng/mL, 10 ng/mL 24 hr,4 hr
    2 HG1 −1.13 HUAEC Cells, Untx, HUAEC Cells, t/TNFa 10 ng/mL
    Nrml 4 hr, Nrml
    2 HG1 −1.14 HUVEC Cells, Untx, HUVEC Cells, t/CHX + TNFa
    Nrml 10 mcg/mL, 10 ng/mL 30 min, 24 hr, Nrml
    2 HG1 −1.14 HUVEC Cells, Untx, HUVEC Cells, t/PMA 10 nM
    Nrml, 24 hr 24 hr, Nrml
    2 HG1 −1.15 HUVEC Cells, Untx, HUVEC Cells, t/TNFa 10 ng/mL
    Nrml 24 hr, Nrml
    2 HG1 −1.17 HUVEC Cells, Untx, HUVEC Cells, t/TNFa 10 ng/mL
    Nrml 24 hr, Nrml
    2 HG1 −1.18 HUVEC Cells, Untx, HUVEC Cells, t/8ClcAMP + TNFa
    Nrml, 24 hr 7.5 microM, 10 ng/mL 24 hr, 4 hr, Nrml
    2 HG1 −1.19 HUVEC Cells, Untx, HUVEC Cells, t/TNFa 10 ng/mL
    Nrml 24 hr, Nrml
    2 HG1 −1.21 HUVEC Cells, Untx, HUVEC Cells, t/TL1b + TNFa
    Nrml 10 ng/mL, 10 ng/mL 2 4hr, 24 hr, Nrml
    2 HG1 −1.22 HIAEC Cells, Untx, HIAEC Cells, t/TNFa 10 ng/mL
    Nrml 24 hr, Nrml
    2 HG1 −1.22 HUVEC Cells, Untx, HUVEC Cells, t/TNFa + TNFa
    Nrml, 24 hr .1 ng/ml, 10 ng/ml 24 hr, 24 hr, Nrml
    2 HG1 −1.23 HUVEC Cells, Untx HUVEC Cells, t/TNFa 10 ng/mL
    4 hr”
    2 HG1 −1.26 HUVEC Cells, Untx, HUVEC Cells, t/8ClcAMP + TNFa
    Nrml, 24 hr 7.5 microM, 10 ng/mL 24 hr, 24 hr, Nrml
    2 HG1 −1.28 HUVEC Cells, Untx HUVEC Cells, t/TNFa .1 ng/mL 4 hr
    2 HG1 −1.29 HUVEC Cells, Untx, HUVEC Cells, t/TNFa 10 ng/mL
    Nrml 4 hr, Nrml
    2 HG1 −1.29 HIAEC Cells, Untx, HIAEC Cells, t/TNFa 10 ng/mL
    Nrml 4 hr, Nrml
    2 HG1 −1.30 HUVEC Cells, Untx HUVEC Cells, t/IL10 + TNFa
    10 ng/mL, 10 ng/mL 24 hr, 24 hr
    2 HG1 −1.31 HAEC Cells, Untx, HAEC Cells, t/TNFa 10 ng/ml
    Nrml 10 hr, Nrml
    2 HG1 −1.31 HAEC Cells, Untx, HAEC Cells, t/TNFa 10 ng/ml
    Nrml 24 hr, Nrml
    2 HG1 −1.34 HUVEC Cells, Untx, HUVEC Cells, t/lL4 + TNFa
    Nrml, 24 hr 10 ng/ml, 10 ng/ml 24 hr, 4 hr, Nrml
    2 HG1 −1.36 HAEC Cells, Untx, HAEC Cells, t/TNFa 10 ng/ml
    Nrml 4 hr, Nrml
    2 HG1 −1.40 HUVEC Cells, Untx, HUVEC Cells, t/IL4 + TNFa
    Nrml, 24 hr 10 ng/ml, 10 ng/ml 24 hr, 24 hr, Nrml
    2 HG1 −1.43 HUVEC Cells, Untx HUVEC Cells, t/TNFa 10 ng/mL 24 hr
    2 HG1 −1.44 HUVEC Cells, Untx, HUVEC Cells, t/IL1b 10 ng/mL
    Nrml 24 hr, Nrml
    2 HG1 −1.48 HUVEC Cells, Untx HUVEC Cells, t/TNFa 10 ng/mL
    24 hr”
    2 HG1 −1.52 HUAEC Cells, Untx, HUAEC Cells, t/TNFa 10 ng/mL
    Nrml 8 hr, Nrml
    2 HG1 −1.53 HUVEC Cells, Untx, HUVEC Cells, t/TNFa 10 ng/mL
    Nrml 4 hr, Nrml
    2 HG1 −1.53 HUVEC Cells, Untx HUVEC Cells, t/CHX + TNFa
    10 mcg/ml, 10 ng/ml 30 min, 23.5 hr
    2 HG1 −1.53 HUVEC Cells, Untx HUVEC Cells, t/TNFa 1 ng/mL 24 hr
    2 HG1 −1.55 HAEC Cells, Untx, HAEC Cells, t/TNFa 10 ng/ml
    Nrml 8 hr, Nrml
    2 HG1 −1.58 HUVEC Cells, Untx, HUVEC Cells, t/PD98059 + TNFa
    Nrml, 24 hr 50 microM, 10 ng/mL 24 hr,24 hr, Nrml
    2 HG1 −1.59 HUVEC Cells, Untx, HUVEC Cells, t/TNFa 10 ng/mL
    Nrml 3 d, Nrml
    2 HG1 −1.59 HAEC Cells, Untx, HAEC Cells, t/TNFa 10 ng/ml
    Nrml 6 hr, Nrml
    2 HG1 −1.61 HUVEC Cells, Untx, HUVEC Cells, t/IL1b + TNFa
    Nrml 10 ng/mL, 10 ng/mL 24 hr, 1 hr, Nrml
    2 HG1 −1.62 HUVEC Cells, Untx, HUVEC Cells, t/TNFa 10 ng/ml
    Nrml, 24 hr 24 hr, Nrml
    2 HG1 −1.72 HUVEC Cells, Untx, HUVEC Cells, t/TNFa 10 ng/mL
    Nrml 8 hr, Nrml
    2 HG1 −1.80 HUVEC Cells, Untx, HUVEC Cells, t/PD98059 + TNFa
    Nrml, 24 hr 50 microM, 10 ng/mL 24 hr, 4 hr, Nrml
    2 HG1 −1.83 HIAEC Cells, Untx, HIAEC Cells, t/TNFa 10 ng/mL
    Nrml 8 hr, Nrml
    2 HG1 −2.07 HUVEC Cells, Untx, HUVEC Cells, t/IL4 + TNFa
    48 hr 10 ng/mL, 10 ng/mL 24 hr, 24 hr
    2 HG1 −2.15 HUVEC Cells, Untx, HUVEC Cells, t/TNFa 10 ng/mL
    Nrml 8 hr, Nrml
    2 HG1 −2.18 HUVEC Cells, Untx, HUVEC Cells, t/IL4 + TNFa
    48 hr 10 ng/mL, 10 ng/mL 24 hr,4 hr
    10 UG1 3.38 HUVEC Cells, Untx, HUVEC Cells, t/CHX + TNFa
    Nrml 10 mcg/mL, 10 ng/mL 30 min, 4 hr, Nrml
    10 UG1 3.16 HUVEC Cells, Untx, HUVEC Cells, t/CHX + TNFa
    Nrml 10 mcg/mL, 10 ng/mL 30 min, 1 hr, Nrml
    10 UG1 2.85 HUAEC Cells, Untx, HUAEC Cells, t/TNFa 10 ng/mL
    Nrml 2 hr, Nrml
    10 UG1 2.68 HPAEC Cells, Untx, HPAEC Cells, t/TNFa 10 ng/mL
    Nrml 1 hr, Nrml
    10 UG1 2.64 HPAEC Cells, Untx, HPAEC Cells, t/TNFa 10 ng/mL
    Nrml 2 hr, Nrml
    10 UG1 2.60 HUVEC Cells, Untx, HUVEC Cells, t/PMA + TNFa
    Nrml, 24 hr 100 nM, 10 ng/mL 24 hr, 1 hr, Nrml
    10 UG1 2.55 HUVEC Cells, Untx, HUVEC Cells, t/PMA + TNFa
    Nrml, 24 hr 100 nM, 10 ng/mL 24 hr, 24 hr, Nrml
    10 UG1 2.47 HUVEC Cells, Untx, HUVEC Cells, t/Dex + TNFa
    0 hr, Nrml 100 nM, 10 ng/mL 24 hr, 1 hr Nrml
    10 UG1 2.40 HUVEC Cells, Untx, HUVEC Cells, t/TNFa 10 ng/mL
    Nrml 48 hr, Nrml
    10 UG1 2.39 HUVEC Cells, Unix, HUVEC Cells, t/Dex + TNFa
    Nrml, 0 hr 100 nM, 10 ng/mL 24 hr, 24 hr Nrml
    10 UG1 2.27 HUVEC Cells, Untx, HUVEC Cells, t/IL10 + TNFa
    Nrml 10 ng/mL, 10 ng/mL 24 hr, 24 hr, Nrml
    10 UG1 2.24 HUAEC Cells, Untx, HUAEC Cells, t/TNFa 10 ng/mL
    Nrml 4 hr, Nrml
    10 UG1 2.18 HUAEC Cells, Untx, HUAEC Cells, t/TNFa 10 ng/mL
    Nrml 1 hr, Nrml
    10 UG1 2.17 HUVEC Cells, Untx, HUVEC Cells, t/Dex + TNFa
    Nrml, 0 hr 10 nM, 10 ng/mL 24 hr, 24 hr Nrml
    10 UG1 2.14 HUVEC Cells, Untx, HUVEC Cells, t/Lipom + TNFa
    24 hr 8 mcg/mL, 10 ng/ml 4 hr, 4 hr, 1 hr
    10 UG1 2.11 HUVEC Cells, Untx, HUVEC Cells, t/ILlb + TNFa
    Nrml 10 ng/mL, 10 ng/mL 24 hr, 24 hr, Nrml
    10 UG1 2.04 HUVEC Cells, Untx, HUVEC Cells, t/IFNg + TNFa
    Nrml 200 ng/mL, 10 ng/mL 24 hr, 24 hr, Nrml
    10 UG1 1.95 HUVEC Cells, Untx, HUVEC Cells, t/Dex + TNFa
    Nrml, 0 hr 10 nM, 10 ng/mL 24 hr, 1 hr Nrml
    10 UG1 1.91 HUVEC Cells, Untx, HUVEC Cells, t/Lipom +
    24 hr asOligo + TNFa 8 mcg/mL,
    100 nM, 50 nM, 10 ng/ml 4 hr, 4 hr, 1 hr
    10 UG1 1.91 HUVEC Cells, Untx, HUVEC Cells, t/PD98059 + TNFa
    Nrml, 24 hr 50 microM, 10 ng/mL 24 hr, 1 hr, Nrml
    10 UG1 1.88 HUVEC Cells, Untx, HUVEC Cells, t/Dex + TNFa
    Nrml, 0 hr 100 nM, 10 ng/mL 24 hr, 4 hr Nrml
    10 UG1 1.88 HUVEC Cells, Untx, HUVEC Cells, t/Dex + TNFa
    Nrml, 0 hr 10 nM, 10 ng/mL 24 hr, 4 hr Nrml
    10 UG1 1.86 HUVEC Cells, Untx, HUVEC Cells, t/IL4 + TNFa
    48 hr 10 ng/mL, 10 ng/mL 24 hr, 24 hr
    10 UG1 1.82 HUVEC Cells, Untx, HUVEC Cells, t/Lipom + TNFa
    24 hr 8 mcg/mL, 10 ng/ml 4 hr, 4 hr, 24 hr
    10 UG1 1.82 HUVEC Cells, Untx, HUVEC Cells, t/IL4 + TNFa
    48 hr 10 ng/mL, 10 ng/mL 24 hr, 1 hr
    10 UG1 1.76 HUVEC Cells, Untx, HUVEC Cells, t/IL1b 10 ng/mL
    Nrml 24 hr, Nrml
    10 UG1 1.74 HUVEC Cells, Untx, HUVEC Cells, t/TNFa 10 ng/mL
    Nrml 24 hr, Nrml
    10 HG3 1.20 HUAEC Cells, Untx, HUAEC Cells, t/TNFa 10 ng/mL
    Nrml 1 hr, Nrml
    10 HG3 1.04 HUAEC Cells, Untx, HUAEC Cells, t/TNFa 10 ng/mL
    Nrml 2 hr, Nrml
    12 HG5 1.02 ECV304 Line, t/TNFa ECV304 Line, Untx, Nrml
    10 ng/mL 3 d, Nrml
    12 HG5 −1.07 HUVEC Cells, Untx HUVEC Cells, t/IL10 + TNFa
    10 ng/mL, 10 ng/mL 24 hr, 4 hr
    12 HG5 −1.16 HUAEC Cells, Untx, HUAEC Cells, t/TNFa 10 ng/mL
    Nrml 8 hr, Nrml
    12 HG5 −1.18 HMVEC Cells, Untx, HMVEC Cells, t/TNFa 10 ng/mL
    Nrml 24 hr, Nrml
    12 HG5 −1.24 HMVEC Cells, Untx, HMVEC Cells, t/TNFa 10 ng/mL
    Nrml 8 hr, Nrml
    12 HG5 −1.50 HIAEC Cells, Untx, HIAEC Cells, t/TNFa 10 ng/mL
    Nrml 8 hr, Nrml
    18 HG2 −2.38 HUVEC Cells, Untx, HUVEC Cells, t/IFNg + TNFa
    Nrml 10 ng/mL, 10 ng/mL 24 hr, 24 hr, Nrml
    32 HG3 −1.13 HUVEC Cells, Untx HUVEC Cells, t/PMA + TNFa
    24 hr, Nrml 10 nM, 10 ng/mL 24 hr, 24 hr, Nrml
    33 LG1 −1.17 Tangier Fibroblast Cells, t/LDL
    Cholesterol, Nrml
    34 UG1 −1.04 ECV304 Line, Untx ECV304 Line, t/PMA + Iono
    1 microM, 1 meg/ml 4 hr
    34 UG1 −1.11 ECV304 Line, Untx ECV304 Line, t/PMA + Iono
    1 microM, 1 meg/ml 5 hr
  • [0221]
  • 0
    SEQUENCE LISTING
    <160> NUMBER OF SEQ ID NOS: 26
    <210> SEQ ID NO 1
    <211> LENGTH: 1334
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <220> FEATURE:
    <221> NAME/KEY: unsure
    <222> LOCATION: 674, 735, 788
    <223> OTHER INFORMATION: a, t, c, g, or other
    <400> SEQUENCE: 1
    aggcctccct ccacctgtct tctcagagca gataatggca agcatggctg ccgtgctcac 60
    ctgggctctg gctcttcttt cagcgttttc ggccacccag gcacggaaag gcttctggga 120
    ctacttcagc cagaccagcg gggacaaagg cagggtggag cagatccatc agcagaagat 180
    ggctcgcgag cccgcgaccc tgaaagacag ccttgagcaa gacctcaaca atatgaacaa 240
    gttcctggaa aagctgaggc ctctgagtgg gagcgaggct cctcggctcc cacaggaccc 300
    ggtgggcatg cggcggcagc tgcaggagga gttggaggag gtgaaggctc gcctccagcc 360
    ctacatggca gaggcgcacg agctggtggg ctggaatttg gagggcttgc ggcagcaact 420
    gaagccctac acgatggatc tgatggagca ggtggccctg cgcgtgcagg agctgcagga 480
    gcagttgcgc gtggtggggg aagacaccaa ggcccagttg ctggggggcg tggacgaggc 540
    ttgggctttg ctgcagggac tgcagagccg cgtggtgcac cacaccggcc gcttcaaaga 600
    gctcttccac ccatacgccg agagcctggt gagcggcatc gggcgccacg tgcaggagct 660
    gcaccgcagt gtgntccgca cgcccccgcc agccccgcgc gcctcagtcg ctgcgtgcag 720
    gtgctctccc ggaantcacg ctcaaggcca aggccctgca cgcacgcatc cagcagaacc 780
    tggaccantg cgcgaagagc tcagcagagc ctttgcaggc actgggactg aggaaggggc 840
    cggcccggac ccccagatgc tctccgagga ggtgcgccag cgacttcagg ctttccgcca 900
    ggacacctac ctgcagatag ctgccttcac tcgcgccatc gaccaggaga ctgaggaggt 960
    ccagcagcag ctggcgccac ctccaccagg ccacagtgcc ttcgccccag agtttcaaca 1020
    aacagacagt ggcaaggttc tgagcaagct gcaggcccgt ctggatgacc tgtgggaaga 1080
    catcactcac agccttcatg accagggcca cagccgtctg ggggacccct gaggatctac 1140
    ctgcccaggc ccattcccag cttcttgtct ggggagcctt ggctctgagc ctctagcatg 1200
    gttcagtcct tgaaagtggc ctgttgggtg gagggtggaa ggtcctgtgc aggacaggga 1260
    ggccaccaaa ggggctgctg tctcctgcat atccagcctc ctgcgactcc ccaatgcagg 1320
    atgcattcat tcac 1334
    <210> SEQ ID NO 2
    <211> LENGTH: 1702
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <400> SEQUENCE: 2
    cgttcccact gcaccctgga gaacgagcct ttgcggggtt tctcctggct gtcctccgac 60
    cccggcggtc tcgaaagcga cacgctgcag tgggtggagg agccccaacg ctcctgcacc 120
    gcgcggagat gcgcggtact ccaggccacc ggtggggtcg agcccgcagg ctggaaggag 180
    atgcgatgcc acctgcgcgc caacggctac ctgtgcaagt accagtttga ggtcttgtgt 240
    cctgcgccgc gccccggggc cgcctctaac ttgagctatc gcgcgccctt ccagctgcac 300
    agcgccgctc tggacttcag tccacctggg accgaggtga gtgcgctctg ccggggacag 360
    ctcccgatct cagttacttg catcgcggac gaaatcggcg ctcgctggga caaactctcg 420
    ggcgatgtgt tgtgtccctg ccccgggagg tacctccgtg ctggcaaatg cgcagagctc 480
    cctaactgcc tagacgactt gggaggcttt gcctgcgaat gtgctacggg cttcgagctg 540
    gggaaggacg gccgctcttg tgtgaccagt ggggaaggac agccgaccct tggggggacc 600
    ggggtgccca ccaggcgccc gccggccact gcaaccagcc ccgtgccgca gagaacatgg 660
    ccaatcaggg tcgacgagaa gctgggagag acaccacttg tccctgaaca agacaattca 720
    gtaacatcta ttcctgagat tcctcgatgg ggatcacaga gcacgatgtc tacccttcaa 780
    atgtcccttc aagccgagtc aaaggccact atcaccccat cagggagcgt gatttccaag 840
    tttaattcta cgacttcctc tgccactcct caggctttcg actcctcctc tgccgtggtc 900
    ttcatatttg tgagcacagc agtagtagtg ttggtgatct tgaccatgac agtactgggg 960
    cttgtcaagc tctgctttca cgaaagcccc tcttcccagc caaggaagga gtctatgggc 1020
    ccgccgggcc tggagagtga tcctgagccc gctgctttgg gctccagttc tgcacattgc 1080
    acaaacaatg gggtgaaagt cggggactgt gatctgcggg acagagcaga gggtgccttg 1140
    ctggcggagt cccctcttgg ctctagtgat gcatagggaa acaggggaca tgggcactcc 1200
    tgtgaacagt ttttcacttt tgatgaaacg gggaaccaag aggaacttac ttgtgtaact 1260
    gacaatttct gcagaaatcc cccttcctct aaattccctt tactccactg aggagctaaa 1320
    tcagaactgc acactccttc cctgatgata gaggaagtgg aagtgccttt aggatggtga 1380
    tactggggga ccgggtagtg ctggggagag atattttctt atgtttattc ggagaatttg 1440
    gagaagtgat tgaacttttc aagacattgg aaacaaatag aacacaatat aatttacatt 1500
    aaaaaataat ttctaccaaa atggaaagga aatgttctat gttgttcagg ctaggagtat 1560
    attggttcga aatcccaggg aaaaaaataa aaataaaaaa ttaaaggatt gttgataaaa 1620
    aaaaaaaaaa aaaaagatct ttaattaagc ggcccaagct tattcccttt agtgagggtt 1680
    aattttagct tgcactggcc ac 1702
    <210> SEQ ID NO 3
    <211> LENGTH: 586
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <220> FEATURE:
    <221> NAME/KEY: unsure
    <222> LOCATION: 48, 66, 560, 574, 577, 580
    <223> OTHER INFORMATION: a, t, c, g, or other
    <400> SEQUENCE: 3
    tcgaggactc cgccaactac agctgcgtct acgtggacct gaagccgnct ttcgggggct 60
    acgcgnccag cgagcgcttg gagctgcacg tggacggacc ccctcccagg cctcagctcc 120
    gggcgacgtg gagtggggcg gtcctggcgg gccgagatgc cgtcctgcgc tgcgagggac 180
    ccatccccga cgtcaccttc gagctgctgc gcgagggcga gacgaaggcc gtgaagacgg 240
    tccgcacccc cggggccgcg gcgaacctcg agctgatctt cgtggggccc cagcacgccg 300
    gcaactacag gtgccgctac cgctcctggg tgccccacac cttcgaatcg gagctcagcg 360
    accctgtgga gctcctggtg gcagaaagct gatgcagccg cgggcccagg gtgctgttgg 420
    tgtcctcaga agtgccgggg attctggact ggctccctcc cctcctgttg cagcacaagg 480
    ccggggtctc tggggggctg gagaagcctc cctcattcct cccaggaatt aataaatgtg 540
    aagagagctc tgtttaaaan aaaaaaaaag aaanaanaan aaccaa 586
    <210> SEQ ID NO 4
    <211> LENGTH: 433
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <400> SEQUENCE: 4
    ctcaagaccc agcagtggga cagccagaca gacggcacga tggcactgag ctcccagatc 60
    tgggccgctt gcctcctgct cctcctcctc ctcgccagcc tgaccagtgg ctctgttttc 120
    ccacaacaga cgggacaact tgcagagctg caaccccagg acagagctgg agccagggcc 180
    agctggatgc ccatgttcca gaggcgaagg aggcgagaca cccacttccc catctgcatt 240
    ttctgctgcg gctgctgtca tcgatcaaag tgtgggatgt gctgcaagac gtagaaccta 300
    cctgccctgc ccccgtcccc tcccttcctt atttattcct gctgccccag aacataggtc 360
    ttggaataaa atggctggtt cttttgtttt ccaaaaaaaa aaaaaaaaaa aaaaaaaaaa 420
    aaaaaaaaaa aaa 433
    <210> SEQ ID NO 5
    <211> LENGTH: 3111
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <220> FEATURE:
    <221> NAME/KEY: unsure
    <222> LOCATION: 44
    <223> OTHER INFORMATION: a, t, c, g, or other
    <400> SEQUENCE: 5
    cgagggcgga cgcaaagaac gcggaggacc tctgggtgcc tgcnggggag ctgctccagc 60
    cgggccgccg ggagcggtgg ggagagcatc gcgcagccgc ccctccacgc gcccgcccag 120
    ccgcgttcgc ccactgggct ctcccggctg cagtgccagg gcgcaggacg cggccgatct 180
    cccgctcccg ccacctccgc caccatgctg ctcccccagc tctgctggct gccgctgctc 240
    gctgggctgc tcccgccggt gcccgctcag aagttctcgg cgctcacgtt tttgagagtg 300
    gatcaagata aagacaagga ttgtagcttg gactgtgcgg gttcgcccca gaaacctctc 360
    tgcgcatctg acggaaggac cttcctttcc cgttgtgaat ttcaacgtgc caagtgcaaa 420
    gatccccagc tagagattgc atatcgagga aactgcaaag acgtgtccag gtgtgtggcc 480
    gaaaggaagt atacccagga gcaagcccgg aaggagtttc agcaagtgtt cattcctgag 540
    tgcaatgacg acggcaccta cagtcaggtc cagtgtcaca gctacacggg atactgctgg 600
    tgcgtcacgc ccaacgggag gcccatcagc ggcactgccg tggcccacaa gacgccccgg 660
    tgcccgggtt ccgtaaatga aaagttaccc caacgcgaag gcacaggaaa aacagatgat 720
    gccgcagctc cagcgttgga gactcagcct caaggagatg aagaagatat tgcatcacgt 780
    taccctaccc tttggactga acaggttaaa agtcggcaga acaaaaccaa taagaattca 840
    gtgtcatcct gtgaccaaga gcaccagtct gccctggagg aagccaagca gcccaagaac 900
    gacaatgtgg tgatccctga gtgtgcgcac ggcggcctct acaagccagt gcagtgccac 960
    ccctccacgg ggtactgctg gtgcgtcctg gtggacacgg ggcgccccat tcccggcaca 1020
    tccacaaggt acgagcagcc gaaatgtgac aacacgggcc agggcccacc cagccaaagc 1080
    ccgggacctg tacaagggcc gccagctaca aggttgtccg ggtgccaaaa agcatgagtt 1140
    tctgaccagc gttctggacg cgctgtccac ggacatggtc cacgccgcct ccgacccctc 1200
    ctcctcgtca ggcaggctct cagaacccga ccccagccat accctagagg agcgggtggt 1260
    gcactggtac ttcaaactac tggataaaaa ctccagtgga gacatcggca aaaaggaaat 1320
    caaacccttc aagaggttcc ttcgcaaaaa atcaaagccc aaaaaatgtg tgaagaagtt 1380
    tgttgaatac tgtgacgtga ataatgacaa atccatctcc gtacaagaac tgatgggctg 1440
    cctgggcgtg gcgaaagagg acggcaaagc ggacaccaag aaacgccaca cccccagagg 1500
    tcatgctgaa agtacgtcta atagacagcc aaggaaacaa ggataaatgg ctcatacccc 1560
    gaaggcagtt cctagacaca tgggaaattt ccctcaccaa agagcaatta agaaaacaaa 1620
    aacagaaaca catagtattt gcactttgta ctttaaatgt aaattcactt tgtagaaatg 1680
    agctatttaa acagactgtt ttaatctgtg aaaatggaga gctggcttca gaaaattaat 1740
    cacataccaa tgtatgtgtc ctcttttgac cttggaaatc tgtatgtggt ggagaagtat 1800
    ttgaatgcat ttaggcttaa tttcttcgcc ttccacatgt taacagtaga gctctatgca 1860
    ctccggctgc aatcgtatgg ctttctctaa cccctgcagt cacttccaga tgcctgtgct 1920
    tacagcattg tggaatcatg ttggaagctc cacatgtcca tggaagtttg tgatgtacgg 1980
    ccgaccctac aggcagttaa catgcatggg ctggtttgtt tcttgggatt ttctgttagt 2040
    ttgtcttgtt ttgctttcca gagatcttgc tcatacaatg aatcacgcaa ccactaaagc 2100
    tatccagtta agtgcaggta gttcccctgg aggaaataat attttcaaac tgtcgttggt 2160
    gtgatacttt ggctcaaagg atctttgctt ttccatttta agcttctgtt ttgagttttg 2220
    ccctggggct tgaatgagtc ccagagagtc gttcggatgg tgggaggctg cctaggaggc 2280
    agtaaatcca gttcacagtg cctgggaggg gcccatcctt ccaaaatgta aatccagttc 2340
    gcggtgtgac cgagctgggc taacaggctt gtctgcctgg ttttcctacc tacacgtgga 2400
    cattattctc ctgatcctcc tacctggttc caccccaggg ctaccggaag gtaaaatctt 2460
    cacctgaacc aattatgagc agtctcctta ctgaaggtac agccggatac gtggtgcccc 2520
    cggggctggt gttggcagcc ggggggaggt gcctgagggt ccccacggtt cctttctgct 2580
    tttctgaatg catcaagggt acgagaactt gccaatggga aattcatccg agtggcactg 2640
    gcagagaagg ataggagtgg aatgcccaca cagtgaccaa cagaactggt ctgcgtgcat 2700
    aaccagctgc caccctcagg cctgggcccc agagctcagg gcacccagtg tcttaaggaa 2760
    ccatttggag gacagtctga gagcaggaac ttcaagctgt gattctatct cggctcagac 2820
    ttttggttgg aaaaagatct tcatggcccc aaatcccctg agacatgcct tgtagaatga 2880
    ttttgtgatg ttgtgatgct tgtggagcat cgcgtaaggc ttcttgctta tttaaactgt 2940
    gcaaggtaaa aatcaagcct ttggagccac agaaccagct caagtacatg ccaatgttgt 3000
    ttaagaaaca gttatgatcc taaacttttt ggataatctt ttatatttct gacctttgaa 3060
    tttaatcatt gttcttagat taaaataaaa tatgctattg aaactaaaaa a 3111
    <210> SEQ ID NO 6
    <211> LENGTH: 2311
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <220> FEATURE:
    <221> NAME/KEY: unsure
    <222> LOCATION: 474-544, 2288, 2295-2299
    <223> OTHER INFORMATION: a, t, c, g, or other
    <400> SEQUENCE: 6
    gccgctcgcc cacggactcc gacgtgtccc tcgactccga ggactccggg gctaagtctc 60
    caggcatcct gggctacaat atctgtcccc gcgggtggaa tggcagcctt cggctcaagc 120
    gtggcagcct ccccgccgag gcctcctgca ccacctagag ccccaccccc gaccccaccc 180
    cgggagggca gagccagaag aaggctcatt agacctgggg gacccaaagg gtctggcctc 240
    tttgggcagc cccagagatg aggggtcagc agaggagagc tctggggttg gggatgggtt 300
    agggacgcaa gcttgagttc tagcccttgc tctcattcag ctgttgtgtg accctgggta 360
    agacccttcc ttgtttgacc ctcagctttc ccatctgttt aatggtggct ttggccaagg 420
    caatccacaa acgtcaaaat tccccttccc atcagtacac acaccgatgc acannnnnnn 480
    nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 540
    nnnntagtta gtgccttgga tgaggcgggg cagtgtgtat atggacccct ggacttgcta 600
    ccttcagggt tccatactcg tccctcccct cctggctctg ctgtctggag tctggcaagc 660
    ggggtgtgtt cagaaggtcc taggcctgtg tcgcatgtcc aggcactggc ctgaccatcc 720
    ggctccctgg gcaccaagtc ccagggcagg agcagctgtt ttccatccct tcccagacaa 780
    gctctatttt tatcacaatg acctttagag aggtctccca ggccagctca aggtgtccca 840
    ctatcccctc tggagggaag aggcaggaaa attctccccg ggtccctgtc atgctacttt 900
    ctccatccca gttcagactg tccaggacat cttatctgca gccataagag aattataagg 960
    cagtgatttc ccttaggccc aggacttggg cctccagctc atctgttcct tctgggccca 1020
    ttcatgggca ggttctgggc tcaaagctga actggggaga gaagagatac agagctacca 1080
    tgtgacttta cctgattgcc ctcagtttgg ggttgcttat tgggaaagag agagacaaag 1140
    agttacttgt tacgggaaat atgaaaagca tggccaggat gcatagagga gattctagca 1200
    ggggacagga ttggctcaga tgacccctga gggctcttcc agtcttgaaa tgcattccat 1260
    gatattagga agtcgggggt gggtggtggt ggtgggctag ttgggcttga atttaggggc 1320
    cgatgagctt gggtacgtga gcagggtgtt aagttagggt ctgcctgtat ttctggtccc 1380
    cttgggaaat gtccccttct tcagtgtcag acctcagtcc cagtgtccat atcgtgccca 1440
    gaaaagtaga cattatcctg ccccatccct tccccagtgc actctgacct agctagtgcc 1500
    tggtgcccag tgacctgggg gagcctggct gcaggccctc actggttccc taaaccttgg 1560
    tggctgtgat tcaggtcccc aggggggact cagggaggaa tatggctgag ttctgtagtt 1620
    tccagagttg ggctggtaga gctttctaga ggttcagaat attagcttca ggatcagctg 1680
    ggggtatgga attggctgag gatcaaacgt atgtaggtga aaggatacca ggatgttgct 1740
    aaaggtgagg gacagtttgg gtttgggact taccggggtg atgttagatc tggaaccccc 1800
    aagtgaggct ggagggagtt aaggtcagta tggaagatag ggttgggaca gggtgctttg 1860
    gaatgaaaga gtgaccttag agggctcctt gggcctcagg aatgctcctg ctgctgtgaa 1920
    gatgagaagg tgctcttact cagttaatga tgagtgacta tatttaccaa agcccctacc 1980
    tgctgctggg tcccttgtag cacaggagac tggggctaag ggcccctccc agggaaggga 2040
    caccatcagg cctctggctg aggcagtagc atagaggatc catttctacc tgcatttccc 2100
    agaggactag caggaggcag ccttgagaaa ccggcagttc ccaagccagc gcctggctgt 2160
    tctctcattg tcactgccct ctccccaacc tctcctctaa cccactagag attgcctgtg 2220
    tcctgcctct tgcctcttgt agaatgcagc tctggccctc aataaatgct tcctgcattc 2280
    taaaaaanaa aaaannnnna aaaaaaaaag g 2311
    <210> SEQ ID NO 7
    <211> LENGTH: 1866
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <400> SEQUENCE: 7
    agcttttgtt cacactttaa atagcagtcc cagaatgatt tcactacaga ctctctggaa 60
    agcctgggag ctgaattccg gaagatcccc acatcgatga aagcaaagcg aagccaccaa 120
    gccatcatca tgtccacgtc gctacgagtc agcccatcca tccatggcta ccacttcgac 180
    acagcctctc gtaagaaagc cgtgggcaac atctttgaaa acacagacca agaatcacta 240
    gaaaggctct tcagaaactc tggagacaag aaagcagagg agagagccaa gatcattttt 300
    gccatagatc aagatgtgga ggagaaaacg cgtgccctga tggccttgaa gaagaggaca 360
    aaagacaagc ttttccagtt tctgaaactg cggaaatatt ccatcaaagt tcactgaaga 420
    gaagaggatg gataaggacg ttatccaaga atggacattc aaagaccaag tgagtttgtg 480
    agattctaac agatgcagca ttttgctgct accttacaag cttctcttct gtcaggactc 540
    cagaggctgg aaagggaccg ggactggaaa gggaccagga ctgaacagac tggttacaaa 600
    gactccaaac aatttcatgc cctgtgctgt tacagaggag aacaaaatgc tttcagcaag 660
    gatttgaaaa ctcttccgtc cctgcaggaa aggattgatg ctgatagaag agcctggaca 720
    gatgtaatga gaactaaaga aaacagatgg ctggagatga catttatcca gggtcacttt 780
    gtcaggccct aggacttaaa tcgaagttga actttttttt ttttttaacc aaatagatag 840
    gggaagggag gagggagagg gaggacaggg agagaaaata ccatgcataa attgtttact 900
    gaatttttat atctgagtgt tcaaaatatt tccaagcctg agtattgtct attggtatag 960
    atttttagaa atcaataatt gattatttat ttgcacttat tacaatgcct gaaaaagtgc 1020
    accacatgga tgttaagtag aaattcaaga aagtaagatg tcttcagcaa ctcagtaaaa 1080
    ccttacgcca ccttttggtt tgtaaaaggt tttttataca tttcaaacag gttgcacaaa 1140
    agttaaaata atggggtctt ttataaatcc aaagtactgt gaaaacattt tacatatttt 1200
    ttaaatcttc tgactaatgc taaaacgtaa tctaattaaa tttcatacag ttactgcagt 1260
    aagcattagg aagtgaatat gatatacaaa atagtttata aagactctat agtttctata 1320
    atttatttta ctggcaaatg tcatgcaaca ataataaatt attgtaaact ttgtggcttt 1380
    tggtctgtga tgcttggtct caaaggaaaa aataagatgg taaatgttga tatttacaaa 1440
    cttttctaaa gatgtgtctc taacaataaa agttaatttt agagtagttt tatattaatt 1500
    accaaacttt ttcaaaacaa attcttacgt caaatatctg ggaagtttct ctgtcccaat 1560
    cttaaaatat aaaatataga tatagaagtt catagattga ctccttggca tttctattta 1620
    tgtatccatt aaggatgagt tttaaaaggc tttctcttca tacttttgaa aaatttcttc 1680
    tatgattaca gtagctatgt acatgtgtac atctattttt cccaagcaat atgttttggg 1740
    tttagagtct gagtgatgac caagattctg tgtgttacta ctgtttgttt aataggaaca 1800
    aatatagaaa taatattatc tctttgctta tttcccgtta aaactataat aaaatgtttc 1860
    taggaa 1866
    <210> SEQ ID NO 8
    <211> LENGTH: 1929
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <400> SEQUENCE: 8
    gctgcctgcc ggtgctcttc gtggctctgg gcatggcctc ggaccccatc ttcacgctgg 60
    cgcccccgct gcattgccac tacggggcct tcccccctaa tgcctctggc tgggagcagc 120
    ctcccaatgc cagcggcgtc agcgtcgcca gcgctgccct agcagccagc gccgccagcc 180
    gtgtcgccac cagtaccgac ccctcgtgca gcggcttcgc cccgccggac ttcaaccatt 240
    gccctcaagg attgggacta taatggcctt cctgtgctca ccaccaacgc catcggccag 300
    tgggatctgg tgtgtgacct gggctggcag gtgatcctgg agcagatcct cttcatcttg 360
    ggctttgcct ccggctacct gttcctgggt taccccgcag acagatttgg ccgtcgcggg 420
    attgtgctgc tgaccttggg gctggtgggc ccctgtggag taggaggggc tgctgcaggc 480
    tcctccacag gcgtcatggc cctccgattc ctcttgggct ttctgcttgc cggtgttgac 540
    ctgggtgtct acctgatgcg cctggagctg tgcgacccaa cccagaggct tcgggtggcc 600
    ctggcagggg agttggtggg ggtgggaggg cacttcctgt tcctgggcct ggcccttgtc 660
    tctaaggatt ggcgattcct acagcgaatg atcaccgctc cctgcatcct cttcctgttt 720
    tatggctggc ctggtttgtt cctggagtcc gcacggtggc tgatagtgaa gcggcagatt 780
    gaggaggctc agtctgtgct gaggatcctg gctgagcgaa accggcccca tgggcagatg 840
    ctgggggagg aggcccagga ggccctgcag gacctggaga atacctgccc tctccctgca 900
    acatcctcct tttcctttgc ttccctcctc aactaccgca acatctggaa aaatctgctt 960
    atcctgggct tcaccaactt cattgcccat gccattcgcc actgctacca gcctgtggga 1020
    ggaggaggga gcccatcgga cttctacctg tgctctctgc tggccagcgg caccgcagcc 1080
    ctggcctgtg tcttcctggg ggtcaccgtg gaccgatttg gccgccgggg catccttctt 1140
    ctctccatga cccttaccgg cattgcttcc ctggtcctgc tgggcctgtg ggattatctg 1200
    aacgaggctg ccatcaccac tttctctgtc cttgggctct tctcctccca agctgccgcc 1260
    atcctcagca ccctccttgc tgctgaggtc atccccacca ctgtccgggg ccgtggcctg 1320
    ggcctgatca tggctctagg ggcgcttgga ggactgagcg gcccggccca gcgcctccac 1380
    atgggccatg gagccttcct gcagcacgtg gtgctggcgg cctgcgccct cctctgcatt 1440
    ctcagcatta tgctgctgcc ggagaccaag cgcaagctcc tgcccgaggt gctccgggac 1500
    ggggagctgt gtcgccggcc ttccctgctg cggcagccac cccctacccg ctgtgaccac 1560
    gtcccgctgc ttgccacccc caaccctgcc ctctgagcgg cctctgagta ccctggcggg 1620
    aggctggccc acacagaaag gtggcaagaa gatcgggaag actgagtagg gaaggcaggg 1680
    ctgcccagaa gtctcagagg cacctcacgc cagccatcgc ggagagctca gagggccgtc 1740
    cccaccctgc ctcctccctg ctgctttgca ttcacttcct tggccagagt caggggacag 1800
    ggagagagct ccacactgta accactgggt ctgggctcca tcctgcgccc aaagacatcc 1860
    acccagacct cattatttct tgctctatca ttctgtttca ataaagacat ttggaataaa 1920
    aaaaaaaaa 1929
    <210> SEQ ID NO 9
    <211> LENGTH: 1831
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <400> SEQUENCE: 9
    ctggagccgc cctgggtgtc agcggctcgg ctcccgcgca cgctccggcc gtcgcgcacc 60
    tcgggcacct gcaggtccgt ggcgtcccgc ggctgggcgc ccctgactcc gtcccggcca 120
    gggagggcca tgatttccct cccggggccc ctggtgacca acttgctgcg gtttttgttc 180
    ctggggctga gtgccctcgc gcccccctcg cgggcccagc tgcaactgca cttgcccgcc 240
    aaccggttgc aggcggtgga gggaggggaa gtggtgcttc cagcgtggta caccttgcac 300
    ggggaggtgt cttcatccca gccatgggag gtgccctttg tgatgtggtt cttcaaacag 360
    aaagaaaagg aggatcaggt gttgtcctac atcaatgggg tcacaacaag caaacctgga 420
    gtatccttgg tctactccat gccctcccgg aacctgtccc tgcggctgga gggtctccag 480
    gagaaagact ctggccccta cagctgctcc gtgaatgtgc aagacaaaca aggcaaatct 540
    aggggccaca gcatcaaaac cttagaactc aatgtactgg ttcctccagc tcctccatcc 600
    tgccgtctcc agggtgtgcc ccatgtgggg gcaaacgtga ccctgagctg ccagtctcca 660
    aggagtaagc ccgctgtcca ataccagtgg gatcggcagc ttccatcctt ccagactttc 720
    tttgcaccag cattagatgt catccgtggg tctttaagcc tcaccaacct ttcgtcttcc 780
    atggctggag tctatgtctg caaggcccac aatgaggtgg gcactgccca atgtaatgtg 840
    acgctggaag tgagcacagg tcagtgaggg ggcctggagc tgcagtggtt gctggagctg 900
    ttgtgggtac cctggttgga ctggggttgc tggctgggct ggtcctcttg taccaccgcc 960
    ggggcaaggc cctggaggag ccagccaatg atatcaagga ggatgccatt gctccccgga 1020
    ccctgccctg gcccaagagc tcagacacaa tctccaagaa tgggaccctt tcctctgtca 1080
    cctccgcacg agccctccgg ccaccccatg gccctcccag gcctggtgca ttgaccccca 1140
    cgcccagtct ctccagccag gccctgccct caccaagact gcccacgaca gatggggccc 1200
    accctcaacc aatatccccc atccctggtg gggtttcttc ctctggcttg agccgcatgg 1260
    gtgctgtgcc tgtgatggtg cctgcccaga gtcaagctgg ctctctggta tgatgacccc 1320
    accactcatt ggctaaagga tttggggtct ctccttccta taagggtcac ctctagcaca 1380
    gaggcctgag tcatgggaaa gagtcacact cctgaccctt agtactctgc ccccacctct 1440
    ctttactgtg ggaaaaccat ctcagtaaga cctaagtgtc caggagacag aaggagaaga 1500
    ggaagtggat ctggaattgg gaggagcctc cacccacccc tgactcctcc ttatgaagcc 1560
    agctgctgaa attagctact caccaagagt gaggggcaga gacttccagt cactgagtct 1620
    cccaggcccc cttgatctgt accccacccc tatctaacac cacccttggc tcccactcca 1680
    gctccctgta ttgatataac ctgtcaggct ggcttggtta ggttttactg gggcagagga 1740
    tagggaatct cttattaaaa ctaacatgaa atatgtgttg ttttcatttg caaatttaaa 1800
    taaagataca taatgtttgt atgagataag a 1831
    <210> SEQ ID NO 10
    <211> LENGTH: 1453
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <220> FEATURE:
    <221> NAME/KEY: unsure
    <222> LOCATION: 903-935
    <223> OTHER INFORMATION: a, t, c, g, or other
    <400> SEQUENCE: 10
    ggagccaagt ggggccctcg gcctcttcct tcgttccagg cccatgattt tccctacact 60
    tctccctggc ccaggctcca gccacaggca cctctcctgc ccccgcccac cctcctgacc 120
    gcagctccca ggccctggag acctccaggc tttcctgccc tgggcagccc cacctcacag 180
    ccagagtcaa tgccttcatg ggaagggctc ccagccacac ccagagtggc ccaaagctgt 240
    tgaagtcagc atcctttgtc ccatcaggac cctcctgcct cctctccagg cccttgttcg 300
    cctccccacc ctcctcagag gcccggggaa gggaagagca ggtcagtaca gaggttctgt 360
    ctacagggag gggccctggg tctatgcaca gctggagctc tgagccttcc acagcccgtg 420
    tgactgctag agggcagggg tgcagggctc aggggggccg ggctggtcct ttggggctgg 480
    tgttcctacg tcagtcccca cctggggaat aaactccagc ctctcctgct catacagaag 540
    gaactggttg ggtttgcttt atgggatctt tgagaccaaa acagatgctc ctgtttgctg 600
    ggggagggtg tgagcacgga gtatttctgt ccctcgtgaa gtcacgtcac acaggggaga 660
    ggcgaggtcg atggaactgg ccacgcacag gctctggctc tggaaggagg gatgatgagt 720
    gggcgttttc ccggcaggcc cccggggtcc tcagcctcag caacccaggg agaggacaga 780
    aatgaaccga tggttgaggg attgtcacgg gaggaacatg acacccgaag ggactctagg 840
    tgccctcgga gtgccacaca tgcccagacc ttctcacacc cacacaaata ggctctgccg 900
    tgnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnttgtt cacactcaga acccaggaca 960
    gccacagcca ccgcttaggg gaagccactg cagatgcccc tggaatgggc acagcacagc 1020
    cagggcgctc ttccaggcag gcgaggataa cttgagagtt tcctagggca ccagggacag 1080
    agctcagagg cccccgaggt gtgtgtagga ggcggaggcc cgcagagcac agagcaggag 1140
    aagggcttgg gccctggagg agaaagccat tctggacacc aggggacctg gacggagggt 1200
    ccccacagcc cgtgccccac gccgcctgga ggccagaggg gtcagtggcc ctgctgtccc 1260
    ggctccatct tggttctagc cgccacctgt atgaacacag tggcccggct taacgcacta 1320
    acccagcctc tccctgtgtc ccacagggag tagcaagacc caccccacac tgccttcacc 1380
    atctacacca gtgacgccgc tgtgtgtctt agcatggaaa taaataaacc tgaatgcaaa 1440
    aaaaaaaaaa agg 1453
    <210> SEQ ID NO 11
    <211> LENGTH: 443
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <400> SEQUENCE: 11
    gatatagaca acttccagag tcaccagtgt gcaaatggag ccacctgcat tagtcatact 60
    aatggctatt cttgcctctg ttttggaaat tttacaggaa aattttgcag acagagcaga 120
    ttaccctcaa cagtctgtgg gaatgagaag acaaatctca cttgctacaa tggaggcaac 180
    tgcacagagt tccagactga attaaaatgt atgtgccggc caggttttac tggagaatgg 240
    tgtgaaaagg acattgatga gtgtgcctct gatccgtgtg tcaatggagg tctgtgccag 300
    gacttactca acaaattcca gtgcctctgt gatgttgcct ttgctggcga gcgctgcgag 360
    gtggacttgg cagatgactt gatctccgac attttcacca ctattggctc agtgactgtc 420
    gccttgttac tgatcctctt gct 443
    <210> SEQ ID NO 12
    <211> LENGTH: 1537
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <220> FEATURE:
    <221> NAME/KEY: unsure
    <222> LOCATION: 284-285, 287
    <223> OTHER INFORMATION: a, t, c, g, or other
    <400> SEQUENCE: 12
    aaaaaaaaca acccggtagc attgtccctt ccccactgac aaacttatca aatccagaag 60
    ctttagagtt tcgtctctaa ttatttttct cctgaacaaa attacccaag tcaaaacaaa 120
    atgtattttt agaattacgg cagcatacga cctgaatttt gtgagtttcg tggctttatc 180
    ttaaatcacc atttccctaa aaacggtttc tttctcctta gaaatgctgg tggcaacttg 240
    atgaaacagc caaatgcacc agggcaggtc actttcccaa aaannanaag aaaaaaaact 300
    cattgagata gctacagttc tataggttaa tttaaagcct cctttttcta ctcatttttg 360
    aaagcaaaat tacattttac tattttacat aaccagtgaa aagacgttga aagcctacag 420
    ctcactgttt ttggtgctct ggaaatgttg agggtgggtt tttaaccagt gatttttaac 480
    gtgcagtgaa tttgttagac ttttaaacac cagctaaggt agtcaaactt gatccccatt 540
    aaaaatcaag gaattagggg tcgggggagg gtttaggagt gatccagaat gacctcccag 600
    aattactgtg cgtacaactt tatttttcag agttttcatt ggaatggtaa gagttttatg 660
    aaagacagtt ttaaaactta ttctgagtta aatattaata ctttaaaaaa ttattgtact 720
    agacttattg cagccttttg aaagtagcag agtttcatca taccacatat ataacagagc 780
    ataaattttc tataatcagg caccttttgc tgcttttgag taagactgtt ttcctgttta 840
    ggtgttaagc atcgccagac ataaaaatct attctctcct ctcgattgta gcatagcctg 900
    acagctctag atacagcatt tctatgatga aaaatgagta tccatcagga aatctagaag 960
    actagccgtg ttttctcaga ctccaccttt gtttgcactc tgttgcctgt gaggagcttt 1020
    ctggcatgtg attatttact tcaaaactag agttccaagc acctacatta attattttat 1080
    attgtgtgca gaatagtata tcttttaatg tcagatatga tacactgcac atattgcttt 1140
    tgcactctta aaatttttgt actaaataat agaaaatatt tatattcttt gagtgtgagc 1200
    tttgaataga tggcattatc actttattgt ttttttaaca aaaacttttt ctcaattatt 1260
    ctattgcaat gttattctga gcaagtccta tgccaaatat cttgtataat gtttgtatgg 1320
    aagattaaat tttactcttg tgtggtaaga ctatttcagt tactgatttt atagttggaa 1380
    tttgatattc cagcacaaag tccacagtgt attcagaaat ccaagttggt gtcatacatt 1440
    tcattttgat gtgaactttt ctttgctttc ctttgttcta agactccatt ttgcaataaa 1500
    cgttttgaca gtaaaaaaaa taaaaaagga aaaaaaa 1537
    <210> SEQ ID NO 13
    <211> LENGTH: 972
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <400> SEQUENCE: 13
    acgcaaattc ggcacgaggg ttctaaaacc cagtttggtt tacgttgtct ttcacagtag 60
    tatatttagc tcttctctgg aaagttgtgg gttaatataa ttcttaaaca tgaaaatgta 120
    attaaacaca ccacgagaga acaatattcc aggagactta atagtgatta ctttcttcaa 180
    tcaggaaatc gtttcagtgc ctcctttgta ggaatgcttt gttttgtgat gggttttctt 240
    aaagaagagc acacctccgt ccaatctcct gagacagcca cgtctccgct gacatcccac 300
    tgtgatgctt tcagatagtc agtgaatgtt tctgataacc ttcatccagt atctgaaaca 360
    caatgtgaga gattatattg ttttagataa taacatccca tttagttgac taaaatcttc 420
    caaactctga aagctgcaca ctgctactcc agagagtgca ggtcttagct cttctccttt 480
    ctgacttcaa gatgaatctt tgggacgatg tttctggtgc ttggtccaca gtgattcact 540
    tttgaaggag aggccacatg acatgaactg cctggtgtta caacctagct aacatatttg 600
    atgctactcc tgttgtctgt actgcttatt caagtagtat tctaagttat gttactaaaa 660
    aacatggtgg gtaaagcaca atcctaccca tcattgtcct ccaaaataat tgtatgacat 720
    acacggccca gcccattgcc ctccctgcat ctctgtgctg ctttgccatt tccccttcta 780
    cccagcctcc tcaaggggta ccttggtgga tatttcagta cttaaaacca gactgtaatc 840
    ataacctccc tctgtgtggc atcaataaat agccaaactc aaaaaaaaaa aaaaaaaaaa 900
    aaaaaaaaaa aaaaaatatc ggtcgcaagc ttattccctt tagtgagggt taattttagc 960
    ttgcactgcc ta 972
    <210> SEQ ID NO 14
    <211> LENGTH: 1544
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <400> SEQUENCE: 14
    tactttgact ttggatcatt tccctgactg ggctaatgtg acacatattg agacttagga 60
    agagccacaa gaccacacac acagccctta ccctcctcag gactaccgaa ccttctggca 120
    caccttgtac agagttttgg ggttcacacc ccaaaatgac ccaacgatgt ccacacacca 180
    ccaaaaccca gccaatgggc cacctcttcc tccaagccca gatgcagaga tggacatggg 240
    cagctggagg gtaggctcag aaatgaaggg aacccctcag tgggctgctg gacccatctt 300
    tcccaagcct tgccattatc tctgtgaggg aggccaggta gccgagggat caggatgcag 360
    gctgctgtac ccgctctgcc tcaagcatcc cccacacagg gctctggttt tcactcgctt 420
    cgtcctagat agtttaaatg ggaatcagat cccctggttg agagctaaga caaccaccta 480
    ccagtgccca tgtcccttcc agctcacctt gagcagcctc agatcatctc tgtcactctg 540
    gaagggacac cccagccagg gacggaatgc ctggtcttga gcaacctccc actgctggag 600
    tgcgagtggg aatcagagcc tcctgaagcc tctgggaact cctcctgtgg ccaccaccaa 660
    aggatgagga atctgagttg ccaacttcag gacgacacct ggcttgccac ccacagtgca 720
    ccacaggcca acctacgccc ttcatcactt ggttctgttt taatcgactg gccccctgtc 780
    ccacctctcc agtgagcctc cttcaactcc ttggtcccct gttgtctggg tcaacatttg 840
    ccgagacgcc ttggctggca ccctctgggg tccccctttt ctcccaggca ggtcatcttt 900
    tctgggagat gcttcccctg ccatccccaa atagctagga tcacactcca agtatgggca 960
    gtgatggcgc tctgggggcc acagtgggct atctaggtcc tccctcacct gaggcccaga 1020
    gtggacacag ctgttaattt ccactggcta tgccacttca gagtctttca tgccagcgtt 1080
    tgagctcctc tgggtaaaat cttccctttg ttgactggcc ttcacagcca tggctggtga 1140
    caacagagga tcgttgagat tgagcagcgc ttggtgatct ctcagcaaac aacccctgcc 1200
    cgtgggccaa tctacttgaa gttactcgga caaagacccc aaagtggggc aacaactcca 1260
    gagaggctgt gggaatcttc agaagccccc ctgtaagaga cagacatgag agacaagcat 1320
    cttctttccc ccgcaagtcc attttatttc cttcttgtgc tgctctggaa gagaggcagt 1380
    agcaaagaga tgagctcctg gatggcattt tccagggcag gagaaagtat gagagcctca 1440
    ggaaacccca tcaaggaccg agtatgtgtc tggttccttg ggtgggacga ttcctgacca 1500
    cactgtccag ctcttgctct cattaaatgc tctgtctccc gcgg 1544
    <210> SEQ ID NO 15
    <211> LENGTH: 1109
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <220> FEATURE:
    <221> NAME/KEY: unsure
    <222> LOCATION: 751-806, 884-885
    <223> OTHER INFORMATION: a, t, c, g, or other
    <400> SEQUENCE: 15
    cggacgcgtg ggggccagcc tggaggccca gacgtggcgc agcgactcgg aggttcgcct 60
    ccagcttgcg catcatctgc ggccgggtcc cgatgagcct cctgttgcct ccgctggcgc 120
    tgctgctgct tctcgcggcg cttgtggccc cagccacagc cgccactgcc taccggccgg 180
    actggaaccg tctgagcggc ctaacccgcg cccgggtaga gacctgcggg ggatgacagc 240
    tgaaccgcct aaaggaggtg aaggctttcg tcacgcagga cattccattc tatcacaacc 300
    tggtgatgaa acacctccct ggggccgacc ctgagctcgt gctgctgggc cgccgctacg 360
    aggaactaga gcgcatccca ctcagtgaaa tgacccgcga agagatcaat gcgctagtgc 420
    aggagctcgg cttctaccgc aaggcggcgc ccgacgcgca ggtgcccccc gagtacgtgt 480
    gggcgcccgc gaagccccca gaggaaactt cggaccacgc tgacctgtag gtccgggggc 540
    gcggcggagc tgggacctac ctgcctgagt cctggagaca gaatgaagcg ctcagcatcc 600
    cgggaatact tctcttgctg agagccgatg cccgtccccg ggccagcagg gatggggttg 660
    gggaggttct cccaacccca ctttcttcct tccccagctc cactaaattc cctcctgcct 720
    taaaaaaaac aagaaaaacc aaacaaacaa nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 780
    nnnnnnnnnn nnnnnnnnnn nnnnnntctt ctatagtgtc acctaaattc aattcactgg 840
    ccgtcgtttt acaacgtcgt gactggcgac ggacaaagtt atcnntttaa tcgccttgca 900
    gcacataccc ctttggccag ctggggtaat aggggaagcg ggccggaccc gatcggcctt 960
    cccaaacagt tggggaagct tgaaatgcgc gacattgggc cgacggcctt ctatacggga 1020
    ggatctctaa acgcggccgg ggtgttggtt gggttaaggc ggagtgtgac cccgcataat 1080
    aacttttgca caggggccct ataggggcc 1109
    <210> SEQ ID NO 16
    <211> LENGTH: 1740
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <400> SEQUENCE: 16
    aagagaagtt accccgatga cttggtttgg aaggggttaa ggcaccagtg catcctcttc 60
    taaagtgatt tatgatgatg tgtggagttt aaaaacttta ccccacccca aagaacagcc 120
    ctctcactcc tcactgagtc cactctgaac gtgctaaaat gggaaggagg cggtgttttg 180
    ctgatctgtt aaattcttag tgaagtttcc ttgatttcca gtggctgctg ttgtttgagt 240
    ttggtttgga gcaaaactga ggtagtccta acatttctgg gactgaatcc aggcaagaga 300
    aagaagaaaa agaagaagaa aaagaggagg aaaaaggtag ggagaaataa agggaggaga 360
    gaagcacagt gaaagaaaaa aaaagtccct tttgcgacat cacattcctg tgttttccct 420
    cagcctggaa aacatattaa tcccagtgct tttacgcccg gaaacaaaga gactaagcca 480
    gactatgggg gaaagggaga taagaaggat cctggaactt taaagaggga aagagtgaga 540
    ttcagaaatc gccaggactg gactttaagg gacgtcctgt gtcagcacaa gggactggca 600
    cacacagaca cacgagaccg aggagaaact gcagacaaat ggagatacaa agacttagaa 660
    ggacagctcc tttcacctca tcctacttgt ccagaaggta aaaagacaca gccagaaaga 720
    aaaggcatcg gctcagctct cagatcagga caggctgtgg atctgtggcg gtactctgaa 780
    agctggagct gcagcacacc ccttttgtat tgctcaccct cggtaaagag agagagggct 840
    gggaggaaaa gtagttcatc taggaaactg tcctgggaac caaacttctg atttcttttg 900
    caaccctctg cattccatct ctatgagcca ccattggatt acacaatgac atggagaatg 960
    ggaccccgtt tcactatgct gttggccatg tggctagtgt gtggatcaga accccacccc 1020
    catgccacta ttagaggcag ccacggagga cggaaagtgc ctttggtttc tccggacagc 1080
    agtaggccag ctcggtttct gaggcacact gggaggtctc gcggaattga gagatccact 1140
    ctggaggaac caaaccttca gcctctccag agaaggagga gtgtgcccgt gttgagacta 1200
    gctcgcccaa cagagccgcc agcccgctcg gacatcaatg gggccgccgt gagacctgag 1260
    caaagaccag cagccagggg ctctccgcgt gagatgatca gagatgaggg gtcctcagct 1320
    cggtcaagaa tgttgcgttt cccttacggg gtccagctct cccaacatcc ttgccagctt 1380
    tgcagggaag aacagagtat gggtcatctc agcccctcat gcctcggaag gctactaccg 1440
    cctcatgatg agcctgctga aggacgatgt gtactgtgag ctggcggaga ggcacatcca 1500
    acagattgtg ctcttccacc aggcaggtga ggaaggaggc aaggtgagaa ggatcaccag 1560
    cgagggccag atcctggagc agcccctggg accctagcct catccctaag ctgatgagct 1620
    tcctgaagct ggagaagggc aagtttggca tggtgctgct gaagaagacg ctgcaggtgg 1680
    aggagcgcta tccatatccc gttaggctgg aagccatgta cgaggtcatc gaccaaggcc 1740
    <210> SEQ ID NO 17
    <211> LENGTH: 4467
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <220> FEATURE:
    <221> NAME/KEY: unsure
    <222> LOCATION: 971, 978, 1295-1522
    <223> OTHER INFORMATION: a, t, c, g, or other
    <400> SEQUENCE: 17
    gcgtcgcgct caccctgcgc gtgcccccgc ggcgcctggg cgtcttcctg gactacgagg 60
    ccggagagct gtccttcttc aacgtgtccg acggctccca catcttcacc ttccacgaca 120
    ccttctcggg cgcgctctgt gcgtacttca ggcccagggc ccacgacggc ggcgaacatc 180
    cggatcccct gaccatctgc ccgctgccgg ttagagggac gcgcgtcccc gaagagaacg 240
    acagtgacac ctggctacag ccctatgagc ccgcggatcc cgccctggac tggtggtgag 300
    gcgccctcgt ggccgcggga ctggccccgg gggggccccc tggatcccag gccagcgctt 360
    tgctctcctg ctccgtctga agggagcagg tgcaccagcc aaaatgtcag cgagggggac 420
    aaagagaggg acctttgcct acgtagatgt gtatgtgtag tgcgattttc ttcaaggaaa 480
    ggagacaagt ccaaagctcg tttgtggatt gtgggactga gcaaaggagt acaaatatat 540
    ccacgtcgct cagagctggg gtgctcacgg tgggtggtgg gaaagaagcc agcatggaag 600
    aaagaaggga gaaaactttg gtgactgcct tagagggatc agttaatttg tatagtttta 660
    tattttttgt atatgtttgc tagctctaaa aaggtcgaga tgcaataaca cttcgtaagc 720
    aacgagttca cctaagtaag gctcagatcc tagttttaaa aaccatttcc cattaaaatg 780
    aagttggagg aacagctgct tctggagccg gggcaaaaaa tttcaaggtg agcctggagc 840
    attgtgtgtg gtgaagtaaa ataaaggctc aaaacgtgac ggcaacccgg caaaagggta 900
    gggagccagg ccgaagggcc tcactgacca attgtgggac aatttgaaca tcaggatgaa 960
    taatgacagg ngaggttnta acacactgaa taaaaacata atccatgagt tcatgctgat 1020
    actcaaattt ctttttaaaa aggagaaaca ggaaggtttc ttttggaggt gaaatctaat 1080
    tattggtgag agtcttggag aacaggctgt ttccagtctc aaagcagtaa ccttatacac 1140
    tacttataag tttgaaaggg gaaaggttac ctttacaatg gagacatcta ccagatgcat 1200
    ccaagtgatt aaatttaaca tcatcaatga tgggaccaag gacattatta gtttgacaac 1260
    tggggaaaga agtgttcttc accccctacc cccannnnnn nnnnnnnnnn nnnnnnnnnn 1320
    nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1380
    nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1440
    nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1500
    nnnnnnnnnn nnnnnnnnnn nnttcatttt cagagtgaga catttgtact gtggctatgt 1560
    aggagaacat tcttgttctt agcaaacata ctgaagtttt tagatattaa ttaccacagt 1620
    gtctgccact gaatttccag tgactaagtg gaaaaatata aaacatatga atataaagaa 1680
    agaaagagac aagtcaaatg tagtaaaatg acaacacttg gtgactctag gtgactggtc 1740
    gacagatgtt cattgtacta tcaatgtggc tttgctgtgg gtttgaaatt ttgcaaacta 1800
    agagttgggt ggcggggaga aggatacacc aaaaaactaa gtgattatct ttggatggga 1860
    aaatgtttgg taattgcatt cttaaaatgt cttctttgta ttttttaatg ttcaataatg 1920
    tatatgtatc agttctgtaa taaaggggaa aacacttttt ttaaatactc ataaaaaacc 1980
    atccgtagga tcgagaagat caggcagaag ggctttgtcc agaaatgtaa ggcctctggt 2040
    gtagagggcc aggtggtggc ggaggggaat gacggtggag ggggagcagg aaggccaagc 2100
    ctgggcagcg agaagaagaa agaggaccca aggagagcac aagtcccacc aaccagagag 2160
    agtcgggtga aggtcctgag aaaactggcc gccactgcac cagctttgcc ccaacctccc 2220
    tcaaccccca gagccaccac ccttcctcct gccccaggcc acaacagtga ctcggtccac 2280
    gtcccgggcg gtaacagttg ctgcaagacc tatgaccacc actgcctttc ccaccacggc 2340
    agaggccctg gaccccctca ccctcccaca ggccccctac aaccactgag gtgatcactg 2400
    ccaggagacc ctcagtttca gagaatcttt accctccatc ccggaaggat cagcacaggg 2460
    agaggccaca gacaaccagg aggcccagca aggccaccag cttggagagc ttcacaaatg 2520
    cccctcccac caccatctca gaacccagca caagggctgc tggcccaggc cgtttccggg 2580
    acaaccgcat ggacaggcgg gaacatggcc accgagaccc aaatgtggtg ccaggtcctc 2640
    ccaagccagc aaaggagaaa cctcccaaaa agaaggccca ggacaaaatt cttagtaatg 2700
    agtatgagga gaagtatgac ctcagccggc ctactgcctc tcagctggag gacgagctgc 2760
    aggtggggaa tgttcccctt aaaaaagcaa aggagtctaa aaagcatgaa aagcttgaga 2820
    aaccagagaa ggagaagaaa aaaaagatga agaatgagaa cgcagacaag ttacttaaga 2880
    gtgaaaagca aatgaagaag tctgagaaaa agagcaagca agagaaagag aagagcaaga 2940
    agaaaaaagg aggtaaaaca gaacaggatg gctatcagaa acccaccaac aaacacttca 3000
    cgcagagtcc caagaagtca gtggccgacc tgctggggtc ctttgaaggc aaacgaagac 3060
    tccttctgat cactgctccc aaggctgaga acaatatgta tgtgcaacaa cgtgatgaat 3120
    atctggaaag tttctgcaag atggctacca ggaaaatctc tgtgatcacc atcttcggcc 3180
    ctgtcaacaa cagcaccatg aaaatcgacc actttcagct agataatgag aagcccatgc 3240
    gagtggtgga tgatgaagac ttggtagacc aagcgtctca tcagcgagct gaggaaagag 3300
    tacggaatga cctacaatga cttcttcatg gtgctaacag atgtggatct gagagtcaag 3360
    caatactatg aggtaccaat aacaatgaag tctgtgtttg atctgatcga tactttccag 3420
    tcccgaatca aagatatgga gaagcagaag aaggagggca ttgtttgcaa agaggacaaa 3480
    aagcagtccc tggagaactt cctatccagg ttccggtgga ggaggaggtt gctggtgatc 3540
    tctgctccta acgatgaaga ctgggcctat tcacagcagc tctctgccct cagtggtcag 3600
    gcgtgcaatt ttggtctgcg ccacataacc attctgaagc ttttaggcgt tggagaggaa 3660
    gttgggggag tgttagaact gttcccaatt aatgggagct ctgttgttga gcgagaagac 3720
    gtaccagccc atttggtgaa agacattcgt aactattttc aagtgagccc ggagtacttc 3780
    tccatgcttc tagtcggaaa agacggaaat gtcaaatcct ggtatccttc cccaatgtgg 3840
    tccatggtga ttgtgtacga tttaattgat tcgatgcaac ttcggagaca ggaaatggcg 3900
    attcagcagt cactggggat gcgctgccca gaagatgagt atgcaggcta tggttaccat 3960
    agttaccacc aaggatacca ggatggttac caggatgact accgtcatca tgagagttat 4020
    caccatggat acccttactg agcagaaata tgtaacctta gactcagcca gtttcctctg 4080
    cagctgctaa aactacatgt ggccagctcc attcttccac actgcgtact acatttcctg 4140
    cctttttctt tcagtgtttt tctaagacta aataaatagc aaactttcac ctattcatga 4200
    gttattattg aaacctcaaa tcataaagac atttaaaaga attgtttttc taactggagg 4260
    ggctctagtg ctaaataata gtactgaaaa ttgatattat tttccttttc ttatatgaag 4320
    gaccttattt ggcatataaa attttataaa atatgtattt aaagcttttt cttatttttt 4380
    gtattaattg gtaagtgaaa actctgttaa agatcacacc acaatgtttt caagaaacat 4440
    ctgaaaagat aaaacaaaga acaaata 4467
    <210> SEQ ID NO 18
    <211> LENGTH: 2965
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <220> FEATURE:
    <221> NAME/KEY: unsure
    <222> LOCATION: 1473-1777, 2948, 2951, 2961
    <223> OTHER INFORMATION: a, t, c, g, or other
    <400> SEQUENCE: 18
    aaacaaagtt caatttagct ggatttctga actatggttt tgaatgttta aagaagaatg 60
    atgggtacag ttaggaaagt ttttttctta cacccgtgac ttgagggaaa cattgcttgt 120
    ctttgagaaa ttgactgaca tactggaaga gaacaccatt ttatctcagg ttagtgaaga 180
    atcagtgcag gtccctgact cttattttcc cagaggccat ggagctgaga ttgagactag 240
    ccttgtggtt ttcacactaa agagtttcct tgttatgggc aacatgcatg acctaatgtc 300
    ttgcaaaatc caatagaagt attgcagctt ccttctctgg ctcaagggct gagttaagtg 360
    aaaggaaaaa cagcacaatg gtgaccactg ataaaggctt tattaggtat atctgaggaa 420
    gtgggtcaca tgaaatgtaa aaagggaatg aggtttttgt tgttttttgg aagtaaaggc 480
    aaacataaat attaccatga tgaattctag tgaaatgacc ccttgacttt gcttttctta 540
    atacagatat ttactgagag gaactatttt tataacacaa gaaaaattta caattgatta 600
    aaagtatcca tgtcttggat acatacgtat ctatagagct ggcatgtaat tcttcctcta 660
    taaagaatag gtataggaaa gactgaataa aaatggaggg atatcccctt ggatttcact 720
    tgcattgtgc aataagcaaa gaagggttga taaaagttct tgatcaaaaa gttcaaagaa 780
    accagaattt tagacagcaa gctaaataaa tattgtaaaa ttgcactata ttaggttaag 840
    tattatttag gtattataat atgctttgta aattttatat tccaaatatt gctcaatatt 900
    tttcatctat taaattaatt tctagtataa ataagtagct tctatatctg tcttagtcta 960
    ttataattgt aaggagtaaa attaaatgaa tagtctgcag gtataaattt gaacaatgca 1020
    tagatgatcg aaaattacgg aaaatcatag ggcagagagg tgtgaagatt catcattatg 1080
    tgaaatttgg atctttctca aatccttgct gaaatttagg atggttctca ctgtttttct 1140
    gtgctgatag taccctttcc aaggtgacct tcagggggat taaccttcct agctcaagca 1200
    aggagctaaa aggagcctta tgcatgatct tcccacatat caaaataact aaaaggcact 1260
    gagtttggca tttttctgcc tgctctgcta agaccttttt ttttttttac tttcattata 1320
    acatattata catgacatta tacaaaaatg attaaaatat attaaaacaa catcaacaat 1380
    ccaggatatt tttctataaa actttttaaa aataattgta tctatatatt caattttaca 1440
    tcctttttca aaggctttgt ttttctaaag gcnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1500
    nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1560
    nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1620
    nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1680
    nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 1740
    nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnatc tgggccttac gtaatatatt 1800
    ttcttaatgg ctgcataata tcacatcaaa taggcatttt tcaaacctct ttccttatta 1860
    aacatgtaga ctatatccat tttttactaa aataaataac atttcagata atatctttgc 1920
    actgataatg ttgccaagcc atttctaaag tgaccttatc aatttaatta ccattggatg 1980
    agggtgttgc tttcatcgca ccattgtaga ttgtcttttt tatttcaatt tgcgtttatt 2040
    tataactggt tgcaaaggta cacagaacac acgctccttc aacttatctt tgataaaccc 2100
    aagcaaggat acaaaaagtt ggacgacatt gagtagagtc atggtatacg gtgctgaccc 2160
    tacagtatca gtggaaaaga taaggaaaat gtcactactc acctatgtta tgcaaaacag 2220
    ttaggtgtgc tggggctgga tactgctctt ttacttgagc attggttgat taaagtttag 2280
    gtaccatcca gggctggtct agagaagtct ttggagttaa ccatgctctt tttgttaaag 2340
    aagagagtaa tgtgtttatc ctggctcata gtccgtcacc gaaaatagaa aatgccatcc 2400
    ataggtaaaa tgctgaccta tagaaaaaaa tgaactctac ttttatagcc tagtaaaaat 2460
    gctctacctg agtagttaaa agcaattcat gaagcctgaa gctaaagagc actctgatgg 2520
    ttttggcata atagctgcat ttccagacct gacctttggc cccaaccaca agtgctccaa 2580
    gccccaccag ctgaccaaag aaagcccaag ttctccttct gtccttccca caacctccct 2640
    gctcccaaaa ctatgaaatt aatttgacca tattaacaca gctgactcct ccagtttact 2700
    taaggtagaa agaatgagtt tacaacagat gaaaataagt gctttgggcg aactgtattc 2760
    cttttaacag atccaaacta ttttacattt aaaaaaaaag ttaaactaaa cttctttact 2820
    gctgatatgt ttcctgtatt ctagaaaaat ttttacactt tcacattatt tttgtacact 2880
    ttccccatgt taagggatga tggcttttat aaatgtgtat tcattaaatg ttactttaaa 2940
    aataaaanaa naaaaaaaaa naaaa 2965
    <210> SEQ ID NO 19
    <211> LENGTH: 1734
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <220> FEATURE:
    <221> NAME/KEY: unsure
    <222> LOCATION: 571-899
    <223> OTHER INFORMATION: a, t, c, g, or other
    <400> SEQUENCE: 19
    cgcctccgga aactgccccc cgggctgctg gccaacttca ccctcctgcg cacccttgac 60
    cttggggaga accagttgga gaccttgcca cctgacctcc tgaggggtcc gctgcaatta 120
    gaacggctac atctagaagg caacaaattg caagtactgg gaaaagatct cctcttgccg 180
    cagccggacc tgcgctacct cttcctgaac ggcaacaagc tggccagggt ggcagccggt 240
    gccttccagg gcctgcggca gctggacatg ctggacctct ccaataactc actggccagc 300
    gtgcccgagg ggctctgggc atccctaggg cagccaaact gggacatgcg ggatggcttc 360
    gacatctccg gcaacccctg gatctgtgac cagaacctga gcgacctcta tcgttggctt 420
    caggcccaaa aagacaagat gttttcccag aatgacacgc gctgtgctgg gcctgaagcc 480
    gtgaagggcc agacgctcct gggcagtggc caagtcccag tgagaccagg ggcttgggtt 540
    gagggtgggg ggtctggtag aacactgcaa nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 600
    nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 660
    nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 720
    nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 780
    nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 840
    nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnna 900
    taatcctgct tttacaggtg aaactcgggg ctgtccatag cggctgggac cccgtttcat 960
    ccatccatgc ttcctagaac acacgatggg ctttccttac ccatgcccaa ggtgtgccct 1020
    ccgtctggaa tgccgttccc tgtttcccag atctcttgaa ctctgggttc tcccagcccc 1080
    ttgtccttcc ttccagctga gccctggcca cactggggct gcctttctct gactctgtct 1140
    tccccaagtc agggggctct ctgagtgcag ggtctgatgc tgagtcccac ttagcttggg 1200
    gtcagaacca aggggtttaa taaataaccc ttgaaaactg gatcggatga attggctttc 1260
    attgtgttcc tagcatcttc tcaaatcaac ttcccaggac tccagggtga aggaggaaaa 1320
    gaggcatggc ccaggccctg gggtgtggga tatggtctcc ctaggggatg acagttggga 1380
    tcaatggcct gtgacttctc ctctcccttc ccccatcctg ggacctaact ggaaataaaa 1440
    ccttgactgt tgcccgggtg tcattttacc agtggatttc tgccagggct tgtgtcctag 1500
    gagaaggttt aagttaaacc agattgccca ggtctccaaa cgatttgtca tgctgacctg 1560
    agatcatcga agggggcacc tgcccccggg caaggttgca ggggcaggat ggggctgaag 1620
    ggatgagcag ggtcccgggc ccacctgctg atacagcatt ggccatgtgg gggctgcaat 1680
    cggatttgga agaccctggg gcttgggggc atgtccattt tcccagtccc taaa 1734
    <210> SEQ ID NO 20
    <211> LENGTH: 4005
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <400> SEQUENCE: 20
    ggacaccgtc tgcagtggag tcactggtgc cgtaaatgtg gccaaggggg ccgtccagac 60
    gggctgtaga cacggccaag accgtgctga ccggcaccaa ggacacagtc actactgggc 120
    tcatgggggc agtgaatgtc gccaaaggga ccgtccagac cagtgtggac accaccaaga 180
    ctgtcctaac tggtaccaag gacaccgtct gcagtggggt gaccggtgct gcgaatgtgg 240
    ccaagggggc cgtccagggg ggcctggaca ctacaaagtc tgtcctgact ggcactaaag 300
    acaccgtatc cactgggctc acaggggctg tgaacttggc caaagggact gtccagaccg 360
    gcgtggacac cagcaagact gtcctgaccg gtaccaagga caccgtctgc agtggagtca 420
    ctggtgccgt aaatgtggcc aaaggcaccg tccagacagg tgtggacaca gccaagacgg 480
    tgctgagtgg cgctaaggat gcagtgacta ctggagtcac gggggcagtg aatgtggcca 540
    aaggaaccgt gcagaccggc gtggacgcct ccaaggctgt gcttatgggt accaaggaca 600
    ctgtcttcag tggggttacc ggtgccatga gcatggccaa aggggccgtc caggggggcc 660
    tggacaccac caagacagtg ctgaccggaa ccaaagacgc agtgtccgct gggctcatgg 720
    ggtcagggaa cgtggcgaca ggggccaccc acactggcct cagcaccttc cagaactggt 780
    tacctagtac ccccgccacc tcctggggtg gactcaccag ttccaggacc acagacaatg 840
    gtggggagca gactgccctg agcccccaag aggccccgtt ctctggcatc tccacgcccc 900
    cggatgtgct cagtgtaggc ccggagcctg cctgggaagc cgcagccact accaagggcc 960
    ttgcgactga cgtggcgacg ttcacccaag gggccgcccc aggcagggag gacacggggc 1020
    ttttggccac cacacacggc cccgaagaag ccccacgctt ggcaatgctg cagaatgagt 1080
    tggaggggct gggggacatc ttccacccca tgaatgcgga ggagcaagct cagctggctg 1140
    cctcccagcc cgggccaaag gtgctgtcgg cggaacaggg gagctacttc gttcgtttag 1200
    gtgacctggg tcccagcttc cgccagcggg catttgaaca cgcggtgagc cacctgcagc 1260
    acggccagtt ccaagccagg gacactctgg cccagctcca ggactgcttc aggctgattg 1320
    aaaaggccca gcaggctcca gaagggcagc cacgtctgga ccagggctca ggtgccagtg 1380
    cggaggacgc tgctgtccag gaggagcggg atgccggggt tctgtccagg gtctgcggcc 1440
    ttctccggca gctgcacacg gcctacagtg gcctggtctc cagcctccag ggcctgcccg 1500
    ccgagctcca gcagccagtg gggcgggcgc ggcacagcct ctgtgagctc tatggcatcg 1560
    tggcctcagc tggctctgta gaggagctgc ccgcagagcg gctggtgcag agccgcgagg 1620
    gtgtgcacca ggcttggcag gggttagagc agctgctgga gggcctacag cacaatcccc 1680
    cgctcagctg gctggtaggg cccttcgcct tgcccgctgg cgggcagtag ctgtaggagc 1740
    ctgcaggccc ggcgcggggt cgccctgctc tgtccaggga ggagctgcct cagaactttc 1800
    tccccgcccc caaacctgga tcggttccct aaagccctag acctttgggg ctgcagctgg 1860
    ctgagcgccg aggggctgcg gaggcagtga ccttcttaac tgagccaccc cacgccctgc 1920
    tccgggcctg cctgcatctc ccacctcctc cccagcgctg cctgcccctc tcggagcctg 1980
    gggtcactca gaccaccagc caagagcctt cccttgaagt ccccaagcaa gcactgcaat 2040
    taggaaagag aaaaagcagc gtgcccagcc tggaagggca tctgtttgcc ccgctagcaa 2100
    cccttttata tctagcaggg ctcttccagt cctgcagcac gggcccccag ctatcagcgg 2160
    tgcaggcagt gctgtggcat cccaggctcc gggcagctcc gttctcatgc tgaaagtggg 2220
    tctccggcct tagcacacac accttgaggg tcttaagaac cacattccct catagtagaa 2280
    agtactagaa aaagcgacac tgccatcatc atcccaaggc aggctgctac tgcctttgct 2340
    gacccccggg gtggcctcac ggtggggaca aagctgccag gagccacagc agccacagct 2400
    ggggctttgc accagcctgg cttgagactg agcagtttgc agggggtggg gggtgcaaaa 2460
    aacaagcaaa caggctgctg ctgcctccag ctgcccacca caggcctgcc ccaggcacct 2520
    ggggctctga ggcccctggg gaggctgggc ccagcagctg cccctggaga acacagacaa 2580
    aggacttccc cgcagggaac tgtgccctat ggagggatca gacagggctg ggaacagcca 2640
    cagaggctgc gtgcctatgg cacagccctt cctccgccgc acactccccc tgggtcctca 2700
    ggcccaccca agcgccgggc tgcagaggaa gcggggctgg ggaggctgca ggcatcagag 2760
    acactggtgg tggcggaccc ggccgccggg ccccgtgctc tcaggctagc ccaggtcgtg 2820
    gaggctggca ggctcaggtc gggtgtgaga cgtgccgtgg ctgcgctcag tccagcgggg 2880
    aggagccgtt cagcccggcc tccccaggaa gccatatccc cactcacccg gtaagagaac 2940
    cttgtcgtcc cctttccatg ctctcctagg acacgagccc aggaacccca gacccagggg 3000
    gaggaagggt ggaggggccc caggggtcac catgtgcacc aggggccgtg aggggccggg 3060
    gcattcagct cagctctgaa ccggggaagc tggcacggca aggactgcct caggtgacgg 3120
    gccgtgagag gggacgggtc aggagccttc ccaagccttc tcctcagccc gacacccatg 3180
    gccatcggag gctaggatgc cagacacagc catttgcaga aatcaggcac agtgactgca 3240
    gctcacgtcc agccaaccaa gcatggggcc gcagctcagg aagtcccttc ccgccacacc 3300
    acagcctaat tcttactggg acggaggcaa ctcggctacg ctgggcagga cgacaaacac 3360
    gagacgccac tgtggaatga gcaacttcgg agcacggggt gacttgcttg ggaccgtgcc 3420
    cacgtgacag ccccttatgc agaggaggaa agagaagccc cgagtgggag gggaacctgt 3480
    ccaaagtcac acggtgtgtg ggtgacacag ctggggtgag tcgaggctgg cccctgaggc 3540
    ccatgctccc tgaacgctgg agaccactgt cggctagcag cggctctcag ggaaggcctg 3600
    gtctccaccc tcccagccta gcctcgcgga ccctcgtcct ccccacatcg gacctgctca 3660
    cctgcctgga ccctgggctg ccagatgcag gaagcatcaa accccccagc ctcgtgggtg 3720
    cggggcaggg cgcaggcagc acagcttaga tgccctggtt tgtccctctt gtctcctggg 3780
    aagagcttgc tcccgcccag ctctcctgcc actggccttt cagggttggg ctgggcccag 3840
    agtgcctttt agtcgcttct cacggtggcc tgatggctca acccagtccc aaacgggccc 3900
    agtgacactg ccgactgcac cccagctcag gcccccactg caccagcaat gctagaaaac 3960
    caagccaata aaagtgattt cttttttcat taaaaaaaaa aaaaa 4005
    <210> SEQ ID NO 21
    <211> LENGTH: 846
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <400> SEQUENCE: 21
    caaaaatgag cggggtgtgg tggcccatgc ctgtagtccc agctgctcgg gagactgaat 60
    ctcttgagcc tgggaagcag aggttgcagt gaactgagat cgcgtcactg cactccagcc 120
    tggtgacaga gcgagattcc atctcaaaaa aaaaaaacag tatgcacgta caaatttctt 180
    aacctgttat caatgtctga gctacataat tatctttcta gttggagttt gttttaggtg 240
    tgtaccaact gacatttcag tttttctgtt tgaagtccaa tgtattagtg actctgtggc 300
    tgctctcttc acctgcccct tgtggcctgt ctacaattct aaatggattt tgaactcaat 360
    gtcgtcgctt ctggtttcct gcatatacca atagcattac ctatgacttt ttttttcctg 420
    agctattttc actgagctga gctaatgaac taaaactgag ttatgtttaa tatttgtatc 480
    aaatacataa aaggaatact gctttttcct tttgtggctc aaaggtagct gcattttaaa 540
    atatttgtga aaataaaaac ttttgttatt agaaaaaaaa aaaaaaaaaa aaaaaaaaaa 600
    aaaaaaaaaa agaaagacca aaaaggaaga gaagggaaaa agaagaagag aaacggaggg 660
    acaacgggaa acacagagag cgagccggtg acgaaaagcg ggaaggccaa cgcaggagaa 720
    gaaagagagg ggggcggcgt cgctcattgt gggagtgtcc tcagagttat gcgagtgggg 780
    gatgatgggc aggagtgcta tgcgcccctt tgtatgaggg ggtgcctcaa ttgttgatgg 840
    gccggg 846
    <210> SEQ ID NO 22
    <211> LENGTH: 1740
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <220> FEATURE:
    <221> NAME/KEY: unsure
    <222> LOCATION: 216-390, 846-889
    <223> OTHER INFORMATION: a, t, c, g, or other
    <400> SEQUENCE: 22
    tacaaagttt taagaaagcc agcatctcag aaaggccttt caaacaagga cacttaatta 60
    gccatcttat gtataagaaa agaaatataa agaacatgaa aatttaaaaa cagatttggc 120
    agttttataa cagtctagga ggtggtgtta ttttttccta ttaagaatta gagggcaggt 180
    taggaataaa taaaatacag tttgaaaata atgagnnnnn nnnnnnnnnn nnnnnnnnnn 240
    nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 300
    nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 360
    nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn gcacttcccc tactgattgc tgccttctct 420
    gtggctacaa gggacccaca gaattacagg gaagttacag ggaagcaggt ttcatctcaa 480
    tattgggaga gatttcaaac aatcacacct gcctgagaag gagtgggctg tcactaggaa 540
    tttttattcc cagtccgtca ggaattttgt agaagggctt catgtgctgg taccaatagg 600
    acaggaagat tttaatcagc tttactatct atgttttttt atggaaactg tgtgtatgta 660
    tacatacatt ttccaaaaag aaaaattaaa tgattataga gattatgttt ttcagactac 720
    tcacgtatct gcttttctta ctccccacct ctgctgataa ttcctagttc attggttttt 780
    cccccacact ggaattacct ggggagctta aaaaaccctg atgcctgggt cccaccctca 840
    gagatnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnt gatatgaatg 900
    cagcctgggc actgggaaat ttaaaaactc cccagataat tactgtgaca gccaaggttg 960
    agacctccta gtctagagct ttgctataca cagggtggtc cacaagccag gagcatcagc 1020
    atcacttggc agcgcttaga gattcagacc ccagacccac tgaatctgac cctgcatttt 1080
    cactagaccc caggtgatca gctgcactag actcaacctc taaaccaaga cctcccaccc 1140
    tcacagtcta tgatccttta gtgaccctca gctgagtcct gtgctgaact gtgtttgttc 1200
    tccttgagca catgcccgct gaccagggac agactggatg agcaagcaac ctgctggcta 1260
    tggagaagag ccaggctggg taaatgtttg ctgtgactaa gccaggatca aagaactgcc 1320
    tgttgcttgc actggctggc actgagcttg ccactctgtg aactgtgctt ccttcccctg 1380
    catggacctg tgcctcagtc actattatcc gcaggccttc tccaagggca gccctctcct 1440
    tgtttatccc tcttaagcct gcgtgcagga aggcacatta accctgtggc cccctgcagg 1500
    caggagggtg ttgggtgccc ttacctacct tgcccttttt cttgtaccgt aggctgtgcc 1560
    gtttatgagt aagtgatgtg tgtctgtgtg tgtgtctaga agtgctgcac tcaccttgtg 1620
    ttattggagg ttgtgtaacc ccctagcttt gagcctggtc tcagatgttc cttttcccgt 1680
    tctctgtcca gccgttaacg cccccagtct gtaataaaag cctatcagcc gtgcacttta 1740
    <210> SEQ ID NO 23
    <211> LENGTH: 499
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <400> SEQUENCE: 23
    ctgggatagc aataacctgt gaaaatgctc ccccggctaa tttgtatcaa tgattatgaa 60
    caacatgcta aatcagtact tccaaagtct aaatatgact attacaggtc tggggcaaat 120
    gatgaagaaa ctttggctga taatattgca gcattttcca gatggaagct gtatccaagg 180
    atgctccgga atgttgctga aacagatctg tcgacttctg ttttaggaca gagggtcagc 240
    atgccaatat gtgtgggggc tacggccatg cagcgcatgg ctcatgtgga cggcgagctt 300
    gccactgtga gagcctgtca gtccctggga acgggcatga tgttgagttc ctgggccacc 360
    tcctcaattg aagaagtggc ggaagctggt cctgaggcac ttcgttggct gctactgtat 420
    atctacaagg accgagaagt caccaagaag ctagtgcggc aggcagagaa gatgggctac 480
    aaggccatat ttgtgacag 499
    <210> SEQ ID NO 24
    <211> LENGTH: 1774
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <220> FEATURE:
    <221> NAME/KEY: unsure
    <222> LOCATION: 1679, 1681, 1684-1686, 1691-1693, 1705-1708, 1710,
    1714, 1716, 1731, 1740, 1744
    <223> OTHER INFORMATION: a, t, c, g, or other
    <400> SEQUENCE: 24
    ctttcctaga caaggctgaa aggggccaac attatttctg aagacttcat tattggaatt 60
    ctatgggagt gatctcactg agctattttg gaatagaaat gtggctagtt gcctgacctc 120
    cctcaatggt ttcacgtggc tttcaaaggg aaggaagggc agtgctgact tttggtaaaa 180
    tgggcgaaag ggtccatgcc agcaacacaa tcactcaaag tccagatgag ggatcagtaa 240
    atacaacgtg cctgaaaggt ggcccttgag cacattcctc cggtagacat taacttatta 300
    aattgattct gattacaaat ataaactttg cccccatctc acccagtaac aatgcaagag 360
    ttgatgtcag tctataaaag gaagtaggaa ctgtccctgg ctttcaggct ccaacatcct 420
    ccccctgtca agatgtggca cctcaaactt tgtgcagtcc tcatgatctt cctgttgctg 480
    ttgggccagg taaggaggga aggatactta tgtgtgtgtg tggagtgtgg agatgatagt 540
    ggtggtggaa cttgaaagct agattcagtc ctgaggaatg gttcctctgt tctgagtcta 600
    cagcatctgc ggaatggaat gatcactctt ccaaggtgtg cagcagggtg tcaacacttt 660
    catatctgaa tgtctttgcc cttacagata gatggctccc caataccaga agtgagttcg 720
    gcaaagagaa ggccacggag aatgacccca ttttggagag gggtttccct caggcctatt 780
    ggagcctcct gccgggatga ttctgagtgt atcacaaggc tatgcagaaa aagacgctgt 840
    tccttaagtg tggcccagga atgatgtaca taccagggaa agaaaggaca gcagtcacct 900
    ccgacaatgc tccgttctat ggaatattga ttaactgcat tttggctgga gacacccaag 960
    tgaagcaatc ttgtattttt aatatttaaa ggcagatgta cgctttaaat tggtctccat 1020
    ttcttcttag aatgttgata tatggataag cataactaaa cttgtcaatt tagagtttat 1080
    ttttctatgg atactattaa atgtctcaaa ttgaaatttt agcagtctgg aattcaagct 1140
    tttgagggaa agaaggattc actttgtata ctaaagaaaa aaacagcatt gcccaataat 1200
    gtgttaactt ctcaatctgg aaagtgtagt gagagctaca taatcaatag ctacgtaatc 1260
    aacttcagca agttcctaag ctgtggccct ggatcccttc actccatact cttcagggag 1320
    gtgtcaaagg tggtcaagct tgggaggctg aggcaggaga atggcgtgaa ccgggagacg 1380
    gacttgcagt gagccgagat ccgccactga ctccagcctg gggaaagagc gagactccgt 1440
    tcacaaaaaa aaagaatcaa aaaaaaaaag gggagccccc ccttggtatc ggaagaccca 1500
    gtcctgtaat tcacacaggt tgagttcaag gcattaagcc ctgtaagggc cacttcggcc 1560
    cctcagagtt gctgttctga tccaacggaa gccgcttaca aatttccctt cggaatttgc 1620
    ctccggcatt ccctaggggc ggtatttgga agcaaagtcc ttttaacagc cagtgtatnc 1680
    naannncggg nnngcccttg cggcnnnngn ccananattg ctccttcttc ncctcttctn 1740
    tttnttcccc cccgtgtcga cagggggtgt ggtc 1774
    <210> SEQ ID NO 25
    <211> LENGTH: 4158
    <212> TYPE: DNA
    <213> ORGANISM: Homo sapiens
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <220> FEATURE:
    <221> NAME/KEY: unsure
    <222> LOCATION: 3667-3722
    <223> OTHER INFORMATION: a, t, c, g, or other
    <400> SEQUENCE: 25
    ctcccacaac aatttcattg ttgttagcat atctatttct ccatacattg taaaactgta 60
    atccttaggt atttctaaaa cataaagagg agaattaagt cagctgcaga acaatggggc 120
    tgattcttct gctttttctc tggaaaatct ttcattgctt ttggtggaaa tttacctaga 180
    ggttacaacc acaggatgta gcttggtctc ttatttgcct ttttgggaaa ccaattaaga 240
    ttaatacagg ataaaggaaa aaagcaatct attcattata taacacagtt gtttgtatta 300
    cttgttccct gcaaaggaaa tctgttgaat gcttgcattt tgaattcttt tctaatagaa 360
    caaccaaaaa aggcttctta tggtgcagca ggaaaaaaga tcatttttat agctttgcat 420
    tcttaacata gcatttaaag agcggcatga attagaggaa agacatggaa cacacaggta 480
    gtcggtttga gatcatcggc ttaaaagtat cctaggatgg taatgaccca gaagtatttc 540
    cagttgtcta gtggtgtggt atgcaggaat gagaagtgtt ttctttccat ttcctgttgg 600
    acaggtggca atcttagcag agccactatt tggagttgat aactaaagat gcaaataacg 660
    tgactatgcc ttctggtcat cctacgacta tttggagttc tccaaaacct tgtaagaggc 720
    atgtcaggca tgcagtaaaa gcatctacaa cttcagctgg gcactggcag cataggtctc 780
    atcttggacc atacagtccc actttataga agagagtgga agttctccaa aacaatatcc 840
    acaacaaagt ctgacctcac tctgagggag atgggaagtg ggaggaagaa ggactaacca 900
    gctccctgga gtaagaggaa tttgctttcc ctgtctgccc accaggggct atatgtgcca 960
    cctttcaggt tggggccaag gaagtgatgt cagtgtgaca gaagggagag ttagacctcc 1020
    agacgtcagc ctccctccca tggggtacat tttcaatctg agtgttgttg ccttagctgt 1080
    gttggtatta gcttgattgg ttggtccgct ggttatgagg tgtagggagg cagtttttgt 1140
    ttagttttta ggactttgcc tcttcctttg tccttagcat aatttctagg cagagcatcc 1200
    acgaagtcgg ttttcattgc cagctcaaga gcgacaatca tttacgagtt cctatgttat 1260
    gttaggtgcc ttatgtatat tatcccaaat ccactgcatg gtttaaatac aggcactgga 1320
    atataaatga aaaaggtcat tacagtcact gactttctgc aggaccttaa acatttctct 1380
    ttccacaagt ttccccttaa tcatgtgtca aacctctctt cctgacggga atgttgtgct 1440
    ataatgaatc tgcataacgc ttgggattct aggaggaagg aaggttccat ggacatgtaa 1500
    gtacagcata ttcccctcag tcttctagga gggcagagtg aatcccagaa ctggtaagat 1560
    tgggaatctg agcattgcca ctttaatctt agaatattta tcattttgac acatcctgtt 1620
    ttttagagag gaaaacaaac acagtttctg cattggtagt gtaaagcata ccttgttagg 1680
    aacgtgtttt gtaagacaca tttgggttgt cattctagag catgtcaaac tttgtacttc 1740
    aaaatatatt tagtatgatt gttagtggta acatatatca aggctttgaa ttaactgttt 1800
    tatttaattt tcacaagaag cacttatttt agccatagga aaaccaatct gagctacaaa 1860
    tagttcttta aaataagccc aggttattta gctattctag aaagtgccga cttctttcaa 1920
    gaagcaggca ttgtaggaca gctgagaatt atcacatagc ctaaattcta gcctggcagc 1980
    aagagtcaca tctgagatgt ccaaaaaaaa aaaaaaaaaa cacctgatct acattgaaag 2040
    ggggtagact aacgtatgtg agaccatttt cctatttgca gttacaaggt taaagaactt 2100
    tgaaggtcat tcggctgcta agaggcatgt cgaacactct gtgtggctct ttcacagtaa 2160
    accctcctaa gagcagaaga cacatggctg ttagtgtctg cgtttagatt taatttctca 2220
    aataaaggcc cttggctgcg tatcatttca tccagttata aactagggct cctgcaagca 2280
    cccccattct aagggtgaat tattgaaatc agttgctatt tgatgagtca caactggccc 2340
    agcaggcagg gcatttgaag tcatggtcat caaaaagaaa tgattgtttt ttgaaaagct 2400
    aaatgcttaa aatgcttcta gagggaagtc gtggggcgtg tgctcattct ctttaaaatc 2460
    agggttgttg agtttgtttt taaacatttt tataagttca tgagaaaaaa tatataaatt 2520
    ctaagaacca acactgtatt cccagaaaca tgaccctcgc tggtcttggg tccacatatc 2580
    attggactct gggggacaca aagatgcctg tgacactttg gtgttgccga gttagtcaac 2640
    aattattctg ggaaaaagca gaattgaatt cttctctaga tgtcctacca gggttggcca 2700
    agggccacaa agcaggctaa taaattccca caggatccag acaccaggca aaattgctct 2760
    aagaagccag ttactgtcat ccctctatgg ttctagaaaa aatagtacaa aaatgacagg 2820
    tcatcctatg agcgtcatgc caatgaaacc ccatcttctg gagaagccct tgaatcagaa 2880
    ttatcttttt tcttgatgtc gtcagatgca gccagtttct taattttttt aaaaactgta 2940
    tgtttctgtg gtatgtatat ttgtacacct aactacctgg cacttggaaa tcacagcact 3000
    actcagaggc aattgaataa agagaaattt aattttaaat atcaagtcct gtcaaacatt 3060
    tctcaaactt ctgattttat caaaggtttg ccagccaata aagtgcatcc caagtataca 3120
    ggggagaaag ctagactcct acagggtcct agagtttaag taattttttt gttattaata 3180
    taggtaataa tttttctaat ttttattttt tggttccaaa tgtaaagctc cttgtgttta 3240
    cctctgttta tgtcattctt gacatgttta tctaaattat gtgtgctctg tgacaggtga 3300
    aatgtaaatc tgggatccat agtcaagata tcataaggac ctacttccca gcctaccttt 3360
    cttcctctac ctgataatga taatactcaa aataacaaca ttcaaaggaa acacaaagaa 3420
    atcctgcttt cacatctcct atttcttggg ctccttaata actactgatg gtttgttcat 3480
    gaaaaaaaat ttttaaatca aaagattgta cttggccctg agttgaaaaa atttcaaaaa 3540
    tcaaaagttt gtacttggcc ctgagttgaa aaaaaaaatt cacattctaa gaataaacag 3600
    aaaaatgttc ttcttggaag taaataacaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3660
    aaaaaannnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 3720
    nntcttctat agtgtcacct aaattcaatt cactggccgt cgttttacaa cgtcgtgact 3780
    gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgcaagct 3840
    ggcgtaatac gcgaagaggc ccgaaccgtt ggcccttccc aacagttgcg cagcctgaat 3900
    ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttagtaga 3960
    ggtgtgccgt aaaaaataga ataatttttt ttcaagagat gagcagaatt gagtaggaat 4020
    gattacgggg aggaaaagat ctagaagata gacaatagag aggagagaaa aagagggacg 4080
    aggaggctga gaggaaaaga gtagaagcgt gatatgaata tatacagaaa cagaaaaagg 4140
    agagagggta agacataa 4158
    <210> SEQ ID NO 26
    <211> LENGTH: 366
    <212> TYPE: PRT
    <213> ORGANISM: Homo sapiens
    <220> FEATURE:
    <221> NAME/KEY: misc_feature
    <400> SEQUENCE: 26
    Met Ala Leu Arg Phe Leu Leu Gly Phe Leu Leu Ala Gly Val Asp
    1 5 10 15
    Leu Gly Val Tyr Leu Met Arg Leu Glu Leu Cys Asp Pro Thr Gln
    20 25 30
    Arg Leu Arg Val Ala Leu Ala Gly Glu Leu Val Gly Val Gly Gly
    35 40 45
    His Phe Leu Phe Leu Gly Leu Ala Leu Val Ser Lys Asp Trp Arg
    50 55 60
    Phe Leu Gln Arg Met Ile Thr Ala Pro Cys Ile Leu Phe Leu Phe
    65 70 75
    Tyr Gly Trp Pro Gly Leu Phe Leu Glu Ser Ala Arg Trp Leu Ile
    80 85 90
    Val Lys Arg Gln Ile Glu Glu Ala Gln Ser Val Leu Arg Ile Leu
    95 100 105
    Ala Glu Arg Asn Arg Pro His Gly Gln Met Leu Gly Glu Glu Ala
    110 115 120
    Gln Glu Ala Leu Gln Asp Leu Glu Asn Thr Cys Pro Leu Pro Ala
    125 130 135
    Thr Ser Ser Phe Ser Phe Ala Ser Leu Leu Asn Tyr Arg Asn Ile
    140 145 150
    Trp Lys Asn Leu Leu Ile Leu Gly Phe Thr Asn Phe Ile Ala His
    155 160 165
    Ala Ile Arg His Cys Tyr Gln Pro Val Gly Gly Gly Gly Ser Pro
    170 175 180
    Ser Asp Phe Tyr Leu Cys Ser Leu Leu Ala Ser Gly Thr Ala Ala
    185 190 195
    Leu Ala Cys Val Phe Leu Gly Val Thr Val Asp Arg Phe Gly Arg
    200 205 210
    Arg Gly Ile Leu Leu Leu Ser Met Thr Leu Thr Gly Ile Ala Ser
    215 220 225
    Leu Val Leu Leu Gly Leu Trp Asp Tyr Leu Asn Glu Ala Ala Ile
    230 235 240
    Thr Thr Phe Ser Val Leu Gly Leu Phe Ser Ser Gln Ala Ala Ala
    245 250 255
    Ile Leu Ser Thr Leu Leu Ala Ala Glu Val Ile Pro Thr Thr Val
    260 265 270
    Arg Gly Arg Gly Leu Gly Leu Ile Met Ala Leu Gly Ala Leu Gly
    275 280 285
    Gly Leu Ser Gly Pro Ala Gln Arg Leu His Met Gly His Gly Ala
    290 295 300
    Phe Leu Gln His Val Val Leu Ala Ala Cys Ala Leu Leu Cys Ile
    305 310 315
    Leu Ser Ile Met Leu Leu Pro Glu Thr Lys Arg Lys Leu Leu Pro
    320 325 330
    Glu Val Leu Arg Asp Gly Glu Leu Cys Arg Arg Pro Ser Leu Leu
    335 340 345
    Arg Gln Pro Pro Pro Thr Arg Cys Asp His Val Pro Leu Leu Ala
    350 355 360
    Thr Pro Asn Pro Ala Leu
    365

Claims (20)

What is claimed is:
1 A combination comprising a plurality of cDNAs wherein the cDNAs are SEQ ID NOs:1-25 and the complements of SEQ ID NOs:1-25.
2. An isolated cDNA comprising a polynucleotide having the nucleic acid sequence of SEQ ID No:8.
3. A method for detecting differential expression of one or more cDNAs in a sample containing nucleic acids, the method comprising:
a) hybridizing the combination of claim 1 with nucleic acids of the sample, thereby forming one or more hybridization complexes;
b) detecting the hybridization complexes; and
c) comparing the hybridization complexes with those of a standard, wherein differences between the standard and sample hybridization complexes indicate differential expression of cDNAs in the sample.
4. The method of claim 3, wherein the nucleic acids of the sample are amplified prior to hybridization.
5. The method of claim 3, wherein the sample is from a subject with a disorder associated with atherosclerosis.
6. The method of claim 3, wherein the sample is from aorta, arteries, arterioles, endothelial cells, plaque, or blood.
7. The method of claim 3 wherein the combination is immobilized on a substrate.
8. The method of claim 3 wherein differential expression is diagnostic of angina pectoris, coronary artery disease, myocardial infarction, hypertension, transient cerebral ischemia, mesenteric ischemia, peripheral vascular disease, renal artery stenosis, or stroke.
9. The method of claim 6 wherein the substrate is a microarray.
10. A method of using cDNAs to screen a sample to identify a ligand which specifically binds a cDNA, the method comprising:
a) combining the combination of claim 1 with the sample under conditions to allow specific binding; and
b) detecting specific binding between each cDNA and at least ligand, thereby identifying a ligand that specifically binds to each cDNA.
11. The method of claim 10 wherein the ligands are DNA molecules, proteins, RNA molecules, or transcription factors.
12. A vector containing the cDNA of claim 2.
13. A host cell containing the vector of claim 12.
14. A method for producing a protein, the method comprising the steps of:
a) culturing the host cell of claim 13 under conditions for expression of protein; and
b) recovering the protein from the host cell culture.
15. A protein comprising a polypeptide having the amino acid of SEQ ID NO:26.
16. A method for using a protein to screen a plurality of molecules or compounds to identify at least one ligand which specifically binds the protein, the method comprising:
a) combining the protein encoded by a cDNA of claim 1 with the plurality of molecules or compounds under conditions to allow specific binding; and
b) detecting specific binding between the protein and a molecule or compound, thereby identifying a ligand which specifically binds the protein.
17. The method of claim 16 wherein the plurality of molecules or compounds is selected from agonists, antagonists, antibodies, DNA molecules, small molecule drugs, immunoglobulins, inhibitors, mimetics, peptide nucleic acids, peptides, pharmaceutical agents, proteins, RNA molecules, and ribozymes.
18. A method of using a protein to produce an antibody, the method comprising:
a) immunizing an animal with the protein encoded by a cDNA of claim 1 under conditions to elicit an antibody response;
b) isolating animal antibodies; and
c) screening the isolated antibodies with the protein, thereby identifying an antibody which specifically binds the protein.
19. A antibody produced by the method of claim 18.
20. A method for using an antibody to detect gene expression in a sample, the method comprising:
a) combining the antibody of claim 19 with a sample under conditions which allow the formation of antibody:protein complexes; and
b) detecting complex formation, wherein complex formation indicates expression of the protein in the sample.
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